Interleukin-4 differentially regulates interleukin-2-mediated and CD2-mediated induction of human lymphokine-activated killer effectors.

Natural killer (NK) cells can be differentiated into lymphokine-activated killer (LAK) effectors following stimulation with interleukin (IL)-2. This induction can be negatively regulated by IL-4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9-1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor-alpha (TNF-alpha) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL-2-induced LAK generation, the anti-CD2-triggered LAK activity was not regulated by IL-4. IL-4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti-CD2 by a mechanism involving, at least in part, an increased TNF-alpha production. Using immobilized monoclonal antibodies against the Fc receptor (Fc gamma RIII or CD16) for NK stimulation, we also observed that the anti-CD16-induced LAK activity was not inhibited by IL-4. These data further point to a pivotal role of TNF-alpha as a regulatory cytokine in anti-CD2-induced LAK generation, and suggest that IL-4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non-MHC-restricted cytotoxicity.     

Lymphokine-Activated Killer Cells in Mouse Bone Marrow Chimaeras The Relationship to Natural Killer Cells and to Alloreactive Cytotoxic T Cells

Spleen cells from mouse bone marrow chimaeras were cultured in vitro in mixed lymphocyte cultures (MLC) or in the presence of interleukin 2 (IL-2) without the added alloantigen. Precursors for the nonspecific cytotoxic cells (in this study: lymphokine-activated killer (LAK) cells) lysing natural killer (NK) cell-sensitive YAC-1 lymphoma could be found 10-12 days after the bone marrow reconstitution, simultaneously with the appearance of the NK activity. The ability of LAK cells to lyse NK-resistant tumour targets as well was demonstrated using the P 815 mastocytoma cell line; reactivity against this target was demonstrable 1 week later than the appearance on YAC-1 lysing cells. Phenotypically LAK cells derived from spleen cell cultures of bone marrow chimaeras did not differ from LAK cells derived from normal spleen cell cultures: precursors resided within the Thy 1-, asialo-GM1+ cell population, and effectors expressed both of these antigens. Splenic NK cells of early bone marrow chimaeras (up to 14-18 days after the bone marrow reconstitution) were Thy 1+ cells, and thus LAK cells of bone marrow chimaeras were not derived from these Thy 1+ NK cells. The treatment of effector cells with anti-Thy 1 antibody plus complement (C) abolished the lytic activity totally. However, these cells were not cytotoxic T cells, since alloreactivity, as an indication of the T-cell cytotoxicity, could not be demonstrated until 4-5 weeks after the bone marrow reconstitution.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

Activated Natural Killer Cells Suppress Myelopoiesis in Mice with Severe Combined Immunodeficiency

The in vivo effect of natural killer (NK) cell activation on aulologous myelopoiesis was studied in an environment deficient of functional Tand B cells. Administration of 3.6-bis[2-(Dimethylamino)-ethoxy]-9H-xanthen-9-one dihydrochloride) Tilorone) or recombinant interleukin-2 (rIL-2) to mice with severe combined immunodeficiency (C. B. -I7 scid/scid) resulted in an increase in YAC-1 lysis by their splenocytes as well as bone marrow cells. Recombinant IL-2 furthermore led to a fivefold increase in the cellularity of the spleen. When assayed against human NK/lymphokine-activated killer (LAK) target, K562 cell line. the IL-2-activated mouse cells exhibited no cytotoxicity across the species barrier. Both agents induced a profound suppression of myelopoietic progenitor cells as measured in a 7-day granulocyte-macrophage colony forming cell (GM-CFC) assay. We conclude that the presence of neither functional T nor B cells is necessary for NK cells to mediate inhibition cf myelopoiesis in the autologous host.     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3-) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3- LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Functionally involved cell surface antigens on murine lymphokine activated killer cells

To investigate the possible involvement of some cell surface structures on lymphoid cells in the functional activity of lymphokine activated killer (LAK) cells, a number of monoclonal antibodies (Mab) against such structures was studied for their ability to inhibit LAK activity in a standard cytotoxicity assay against the natural killer-insensitive target cell EL-4. Almost complete inhibition of LAK activity resulted from incubation with antibodies to the LFA-1 antigen, while blocking of the Lyt 2 antigen reduced cytotoxic activity about 50%. Mab to T-200 gave a weak and inconsistent inhibitory activity, while antibodies to Thy 1, L3T4, IL-2 receptor and MHC class I antigens were without effect. Mab to LFA-1 and Lyt 2 inhibited LAK activity towards EL-4, YAC-1 and differentiated F-9 teratocarcinoma cells, but did not affect LAK-mediated killing of undifferentiated F-9 cells. Experiments with separate preincubation of effector and target cells revealed that both LFA-1 and Lyt 2 inhibited LAK activity at the effector cell level only.     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3⁻) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3⁻ LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Up-regulation of induction of lymphokine (IL-2)-activated killer (LAK) cell activity by FK-565 and cisplatin

The role of cisplatin and FK-565 in up-regulation of lymphokine-activated killer (LAK) cell induction by IL-2 was examined. Treatment of blood mononuclear cells (MNC) of healthy donors with cisplatin or FK-565 in the presence of IL-2 resulted in a significant increase in LAK activity against natural killer (NK)-resistant Daudi cells as assessed by the 4 h 51Cr release assay. Blood MNC treated with cisplatin alone was not cytotoxic to Daudi cells. However, MNC treated with FK-565 showed some cytotoxicity against Daudi cells. Addition of cisplatin to IL-2-stimulated MNC did not increase proliferation but did enhance cytotoxicity. FK-565 together with IL-2 increased both proliferation and cytotoxicity of blood MNC. These data suggest the potential of cisplatin and FK-565 in LAK adoptive immunotherapy for cancer treatment.     

Differential effects of dibutyryl cyclic adenosine monophosphate and simple sugars on NK and LAK activities suggesting differences of their cytotoxic mechanism.

The increase of intracellular cyclic AMP levels suppresses the cytotoxic activities of lymphocytes and monosaccharides interfere with cell to cell and cell to cytokine interactions, and these effects on natural killer (NK)/antibody-dependent cell-mediated cytotoxicity (ADCC) and lymphokine activated killer (LAK) activities were examined. Dibutyryl cyclic AMP markedly suppressed NK, IL-2-augmented NK and ADCC activities in a dose-dependent manner but not previously induced LAK activity. Induction of LAK was inhibited. Dibutyryl cyclic GMP had no effect. Addition of mannose 6-phosphate and galactose 6-phosphate strongly inhibited NK and ADCC activities, but not LAK activity. These results suggest that the lytic mechanism for NK and ADCC activity is different from that of LAK activity.     

Interleukin-2 induced killer cell activity against Epstein-Barr virus-immortalized human B cells

Interleukin-2 (IL-2) activated killer (LAK) cells, generated in vitro by treating peripheral blood lymphocytes (PBL) with human IL-2, are able to lyse a wide variety of target cells without restriction by major histocompatibility complex (MHC) molecules. Earlier observations from this and other laboratories indicated that patients with Epstein-Barr virus (EBV) induced infectious mononucleosis, a self-limiting viral disease, have high EBV-nonspecific natural killer (NK) cell activity. Since the effect of LAK cells on EBV-immortalized B lymphocytes has not yet been studied, we decided to investigate LAK cell activity against autologous and heterologous B lymphocytes immortralized in vitro by EBV and other EBV genome-positive and -negative targets of malignant origin. LAK activity was determined by ⁵¹Chromium release assay. The results obtained show that LAK activity was not specific for EBV and was not MHC-restricted. Results of experiments using NK cell reactive monoclonal antibodies suggest that the cytotoxicity is due predominantly to activated NK cells. Our observations suggest that LAK cells may be very effective for immunotherapy in patients with chronic or progressive EBV infections and EBV-induced lymphoproliferative diseases.     

Gelatin sponge model of effector recruitment: tumoricidal activity of adherent and non-adherent lymphokine-activated killer cells after culture in interleukin-2

This study examined the specific tumoricidal activity of lymphokine-activated killer (LAK) cells derived from tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors. A pre-implanted gelatin sponge was employed to capture infiltrating host effectors during the expression of concomitant tumor immunity. Additionally, this study compared the cytolytic activity of these sponge-derived cells with those of counterpart splenic lymphocytes. The cells from both sources were cultured for 4 days in IL-2 to generate LAK cells which were further expanded in IL-2-containing medium for up to 11 days. The cytotoxic activities of these cells were measured in a Chromium-51 release assay. The data revealed that the culture of splenic, or sponge-derived lymphocytes results in the emergence of non-adherent and adherent cell populations with LAK activity. The 4-day sponge-derived LAK cells (adherent and non-adherent) exhibited significant cytolysis of EMT6 cells while the spleen-derived counterparts showed minimal cytotoxicity toward these targets. Some NK activity in LAK cells derived from both sources was evident by their lysis of YAC-1 cells. LAK cells from both sources were incapable of lysing histo-compatible EL-4 (H-2b) tumor cells. The lysis of the EMT6 cells by the sponge-derived LAK cells was maintained over an 11-day period of culture in IL-2. Conversely, the spleen-derived LAK cells were unable to significantly lyse EMT6 cells during this period of in vitro culture. These results show the superior specific tumoricidal activity of LAK cells derived from lymphocytes mediating tumor rejection in vivo (sponge-derived) over that of counterpart splenic lymphocytes.     

Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma.

Natural killer (NK) and lymphokine-activated killer (LAK) cell activities were measured in peripheral blood obtained from healthy women to compare a standard 51Cr release assay with a nonradioactive europium (Eu3+) release assay based on time-resolved fluorescence. The two types of cytotoxicity assays were first compared in paired determinations performed on 28 samples of peripheral blood mononuclear cells obtained from healthy women who had normal pap smears or no biopsy evidence of cervical squamous intraepithelial lesions (SIL). Target cells (NK-sensitive K562 and NK-resistant Raji cell lines) were labeled with Eu3+ only, 51Cr only, or both labels and compared in cytotoxicity assays using fresh or interleukin 2 (IL-2)-activated effector cells. Spontaneous release in the Eu3+ release assay was comparable to that observed in the 51Cr release assay, but maximum Eu3+ release always exceeded that of 51Cr. In 4-h assays, specific release of Eu3+ from target cells was more rapid than that of 51Cr, consistently resulting in 30 to 40% higher levels of activity. However, a significant linear correlation (P < 0.001) was observed between cytotoxicity levels based on measurements of Eu3+ and 51Cr release in 4-h assays. The Eu3+ release assay was then used to measure NK and LAK activities in the peripheral blood of women with cervical SIL or cervical squamous cell carcinoma (SCC). Mean NK activity of women with advanced SIL (121 lytic units [LU]) or SCC (93 LU) was found to be similar to that of controls (101 LU) or patients with normal cervical biopsies (90 LU), as was the ability to generate IL-2-stimulated NK activity. However, LAK activity during 18 h of incubation in the presence of IL-2 was reduced in patients with cervical SCC (P < 0.05) compared with that in normal controls. Results of 51Cr assays performed in parallel with patient samples gave comparable results. Advantages of EU3+ release assays for routine evaluation of cytotoxicity are discussed.     

Lymphokine-activated killer cells in primary immunodeficiencies and acquired immunodeficiency syndrome

Cytotoxic mechanisms (e.g., natural killer (NK) lysis, antibody-dependent cellular cytotoxicity, and cytotoxic T lymphocyte lysis) play an important role in host defense against various infections and neoplasms. Lymphokine-activated killer (LAK) cytotoxicity, induced in vitro by incubating mononuclear cells with interleukin 2 (IL-2) for 2-5 days, may also represent an important component of the body's cytotoxic repertoire. In 10 patients with congenital cellular immunodeficiencies, including 5 with severe combined immunodeficiency, the mean LAK activity in a 3-hr chromium release assay against Raji target cells was 44 +/- 8.1%, which is equivalent to that observed in normal adults and neonates. In only one case, a patient with reticular dysgenesis, was there absent LAK cell generation. Haploidentical T cell-depleted bone marrow transplantation (BMT) restored LAK activity in this patient. LAK activity was first observed in this patient and two others 3-6 weeks following BMT, prior to other evidence of immunologic engraftment such as lymphocyte proliferation to mitogens, NK activity, or interferon-gamma production. One patient with adenosine deaminase deficiency showed normal levels of LAK activity despite absent NK activity. Three patients with chronic granulomatous disease also had normal LAK activity (57 +/- 14% specific lysis). In 9 patients with acquired immunodeficiency syndrome (AIDS), IL-2 activation resulted in a mean cytotoxic activity of 56 +/- 8.7% toward Raji targets. In addition, 9 patients with pre-AIDS complex also showed normal levels of cytotoxicity (37 +/- 3.3% toward Raji targets), equivalent to that of 8 normal controls, including two healthy homosexual males (mean lysis 38 +/- 3.9%). These results indicate that LAK cells appear early in immunologic ontogeny. Further, the mechanism of lysis is not oxygen dependent since LAK activity was present in the 3 patients with chronic granulomatous disease. The ability to generate LAK in a wide spectrum of immunodeficiencies may indicate that IL-2 could be used in therapy of such disorders.     

Cellular cytotoxicity of human natural killer cells and lymphokine-activated killer cells against bladder carcinoma cell lines

The cytotoxicity of natural killer (NK) and lymphokine activated killer (LAK) cells against two human bladder tumor cell lines (BT-A and BT-B) was investigated using a fluorometric assay by labeling tumor cell DNA with Hoechst dye No. 33342. Our results demonstrate that BT-A and BT-B cells have low sensitivity to the cytotoxic activity of mononuclear cells (MNC) and NK cells. Cytotoxicity of MNC or NK cells against both tumor cell lines is enhanced during co-culture of the effector cells with the target cells, which suggests that BT-A and BT-B cells provide the signals which could activate MNC to exert cytotoxicity. In contrast to NK cells, IL-2-generated LAK cells showed profound cytotoxicity to BT-A and BT-B within 24 h. In addition to cellular cytotoxicity to bladder tumor cells, we also tested the effect of recombinant interleukin 1 beta (rIL-1 beta), recombinant tumor necrosis factor (rTNF), and the supernatants of co-culture of MNC or LAK cells with bladder tumor cells. The results show no cytotoxic or growth-promoting activity of rIL-1, rTNF, or the crude culture supernatants on bladder tumor cells. We found that LAK cells, but not macrophages or NK cells, may play a major role in cellular cytotoxicity against the two bladder tumor cell lines tested. From this finding we conclude that activation of LAK cells may be one important mechanism induced by adjuvant bacillus Calmette-Guérin (BCG) therapy leading to effective prevention of urothelial bladder carcinoma reappearance.     

Interleukin-2-activated human effector lymphocytes mediate cytotoxicity by inducing apoptosis in human leukaemia and solid tumour target cells.

The mode of cytotoxic action employed by cytolytic lymphocytes remains unclear, with the possibility of several mechanisms being utilized dependent upon the activation state of the effector cell. In this work, the induction of apoptosis in target cells by 'killer' lymphocytes at differing states of activation has been studied. Although the cytotoxicity of natural killer (NK) cells and recombinant human interleukin-2 (rhIL-2) or interferon-alpha (IFN-alpha)-activated effector cells, against NK-sensitive target cells, was high, their cytotoxic action appeared to be mediated via differing pathways. Effector cells activated short term (4 hr) with rhIL-2 and those mediating rhIL-2 lymphokine-activated killer (LAK) activity after long-term (4 day) activation were found to induce the formation of sodium dodecyl sulphate (SDS)-insoluble apoptotic bodies in NK-sensitive target cells, as well as increasing the level of activity of the apoptosis related enzyme tissue transglutaminase, thus suggesting the induction of the apoptotic pathway as a means of effecting target cell death. Non-activated and short-term (4 hr) IFN-alpha-activated effector cells did not appear to utilize this pathway in the target cell as their means of cytotoxicity. Effector cells showing LAK activity were also cytotoxic towards NK-insensitive cells, and this cytotoxicity again appeared to be mediated via the apoptotic pathway.     

脐带血来源的树突细胞增强LAK细胞的增殖和杀伤活性

目的探讨脐带血来源的树突细胞(CBDC)在体外对LAK(lymphokine activated killer)细胞增殖活性和杀伤活性的影响。方法采用GM-CSF和IL-4联合刺激诱导脐带血单个核细胞分化为CBDC,再将CBDC与同源LAK细胞混合培养,用MTT法测定不同细胞密度的CBDC刺激LAK细胞增殖和杀伤的能力。结果体外诱导出形态典型的CBDC,其具有明显刺激LAK细胞增殖的能力。LAK细胞的增殖和杀伤活性在2种细胞混合比为1∶10时最强。结论CBDC能增强LAK的增殖活性和杀伤活性。     

Differentiation of Macrophage Precursors to Cells with LAK Activity under the Influence of CSF-1 and High Dose IL-2

Mouse macrophage precursor cells with natural killer (NK) like activity, derived from in vitro culture of light-fraction-bone marrow cells in the presence of colony-stimulating factor 1 (CSF-1) and low dose IL-2, were incubated with high dose (1000 U/ml) IL-2. After 3 days of incubation, cells had developed from NK like (killing Yac-1 but not P815) into LAK-like (killing both YAC-1 and P815) effector cells. Morphological studies revealed that LAK activity occurred at the time when macrophage precursors with NK like activity containing few cytoplasmic granules had further differentiated into cells with abundant azurophilic granules in their cytoplasma. Proliferation of macrophage-precursor derived NK/LAK-like cells was dependent on the presence of colony-stimulating factor, generation of cytoplasmic granules was induced by IL-2 in a dose dependent way. Flow cytometric analysis showed that macrophage precursor-derived LAK effectors were positive for NK 1.1 but almost negative for F4/80. When the same starting cell population was cultured in the presence of 200 U/ml Interferon gamma (IFN gamma), proliferation was completely stopped and within 3-4 days all cells differentiated into mature macrophages expressing F4/80. In context with our previous data, we describe here a continuum of development from agranular macrophage precursors to granular cells with NK-like activity and further to cells with LAK activity under the influence of CSF-1 as growth factor and IL-2 as granule- and cytotoxicity-inducing factor.