Susceptibility of adult acute lymphoblastic leukemia blasts to lysis by lymphokine-activated killer cells

To help understand host-tumor relationships in adult acute lymphoblastic leukemia (ALL) and to better define potential indications for interleukin-2 (IL-2) treatment in this disease, the relationship between the susceptibility of leukemia cells of 22 patients with ALL to lysis by allogeneic lymphokine-activated killer (LAK) cells and characteristics of the leukemia was studied. Lymphocytes were activated in the presence of 1000 U/ml recombinant IL-2 for 5 days. The lysis of ALL cells was studied by the release of 51Cr. The average lysis of ALL cells by control, unactivated lymphocytes was 1.2 +/- 2.4% and by LAK cells 8.9 +/- 8.6%. The susceptibility of leukemic cells to lysis did not correlate with the expression of lymphoid or myeloid differentiation markers or expression of the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3). Leukemic cells of the FAB I2 subtype were significantly more resistant to lysis than those of the other subtypes (average lysis 1.4 +/- 3.0% versus 12.3 +/- 8.2%, p = 0.003). The susceptibility to lysis did not correlate with the other initial characteristics of the leukemia. The 11 patients in whom 8% or more of leukemic cells were lysed by allogeneic LAK cells survived significantly longer than the 11 patients whose blast cells were less susceptible to lysis (p = 0.04). It is concluded that IL-2 treatment might be of benefit in adult ALL, particularly in non-L2 FAB subtypes and during complete remission to possibly delay relapse and prolong survival.     

Interferon-gamma-induced alterations of monocyte susceptibility to lysis by autologous lymphokine-activated killer (LAK) cells

Interleukin-2 (IL-2)-activated killer (LAK) cells specifically lyse human monocytes, which may account for some of the toxicity seen during LAK/IL-2 immunotherapy of cancer patients. In an effort to protect autologous monocytes, we treated monolayer cultures of monocytes with various doses of recombinant human interferon-gamma (IFN-gamma) and assessed their sensitivity to LAK-mediated lysis. IFN-gamma lessens the sensitivity of monocytes to lysis in a dose-dependent manner. Treatment of FMEX, an NK-resistant melanoma tumor cell line, with IFN-gamma did not affect its susceptibility to LAK lysis. Kinetic studies demonstrated that as little as 2 hr incubation with IFN-gamma was sufficient for protection to occur, and that monocytes which were treated with IFN-gamma for 2 hr, washed, and then cultured in medium alone retained their resistance to lysis for at least 4 days. Cold target inhibition studies showed that IFN-treated and untreated monocytes could effectively compete with each other for binding sites on LAK cells. Finally, binding studies demonstrated that there was no significant difference between the number of conjugates formed using either IFN-treated or untreated monocytes. This indicates that resistance to lysis induced by IFN treatment affects a post-binding event and not an initial recognition signal.     

Susceptibility to lysis of pulmonary alveolar macrophages by human lymphokine-activated killer cells

In this study we addressed the question of whether lymphokine-activated killer (LAK) cells, besides killing neoplastic cells, may exert a certain degree of lysis on the normal counterpart; in particular we took into consideration the toxicity against pulmonary alveolar macrophages (PAM). We demonstrated that human LAK cells generated in vitro following incubation of peripheral blood mononuclear cells with recombinant interleukin 2 for 4 days were able to lyse normal PAM in a 4-h 51Cr release assay. Similarly, PAM recovered from patients suffering from nonneoplastic interstitial lung disorders, i.e., sarcoidosis and hypersensitivity pneumonitis, were shown to be susceptible to the cytotoxic function provided by LAK cells. Both autologous and allogeneic PAM were lysed by LAK cells, thus suggesting that the phenomenon we observed does not require a major histocompatibility complex restriction. Preincubation of PAM under study with gamma-interferon did not affect their susceptibility to the lysis mediated by LAK cells. Furthermore, cold target inhibition assay demonstrated that normal PAM could efficiently compete with both NK-sensitive and NK-resistant target lines for the binding sites on LAK cells, thus indicating that the putative receptor(s), or at least the mechanism of target recognition, is shared by PAM and these different target cell lines. The evidence herein provided that LAK cells are cytotoxic to normal, nontransformed PAM points out that the pathogenetic mechanisms involving this self-addressed lytic activity could account for some adverse reactions related to LAK/interleukin 2 immunotherapy.     

Morphologic analysis of human lymphokine-activated killer (LAK) cells

Lymphokine-activated killer (LAK) cells obtained from normal donors at various days of in vitro cultivation have been studied by several methods including scanning (SEM) and transmission (TEM) electron microscopy, immuno-electron microscopy, in situ hybridization and flow cytometric DNA measurements. In addition, the cytotoxic activity of LAK cells against several tumor cells was examined by 51Cr-release assay and by SEM and TEM. The LAK cells displayed a uniform ultrastructural appearance concerning surface structure and morphology of organelles. They contained typical lysosomal granules which by immuno-electron microscopy showed a specific localization of perforin I (PI). The presence of PI and granzymeA mRNA in the cytoplasm was confirmed by in situ hybridization using specific antisense probes. Frequency and increased of specific mRNA-containing cells was similar for both genes. Single LAK cells were further characterized by peculiar nuclear inclusion bodies (IB) which were presumably formed by trapped profiles of endoplasmic reticulum. Flow cytometric analysis revealed normal DNA content of LAK cells even after prolonged cultivation indicating that the IB were not associated with aneuploidy of the effector cells. The LAK cells were highly effective in lysing K562 and DAUDI cells as shown by 51Cr-release assay. They caused characteristic morphologic alterations of target cells similar to those found in cytotoxic T-lymphocyte (CTL) and NK-cell-mediated cytolysis. SEM and TEM studies on specimens prepared by routine procedures or by cryopreparation showed that the tumor cell membrane was the initial target for the LAK cell attack whereas other cell compartments were damaged only in advanced stages of cytolysis. Summarizing our study demonstrates that LAK cells have a characteristic ultrastructure which in some aspects differs from that of CTL and NK cells, and that LAK cells appear to destroy tumor cells by mechanisms similar to those of other cytotoxic effector cells.     

Antimetastasis effect of lymphokine-activated killer (LAK) cells on fibrosarcoma in WKA rats

Treating WKA rats with fibrosarcoma with LAK cells, antimetastasis effect was studied. The results indicated that splenocytes incubated by IL-2 could produce lytic activity to WKA rats with fibrosarcoma in vitro. Passive infusion of LAK cells lead to a marked reduction in the number of metastatic nodules in the lung. After intravenous injection of LAK cells, cancer cells in the lung speedily disappeared. LAK cell administration for 3 times gave better result than once only (P less than 0.01). In this paper, the possibility of LAK cells used as an adoptive immunotherapy for human neoplasms is briefly discussed.     

Cytotoxicity of interleukin 2-induced lymphokine-activated killer (LAK) cells against human leukemia and augmentation of killing by interferons and tumor necrosis factor.

Adoptive immunotherapy with interleukin 2-induced lymphokine-activated killer (LAK) cells or tumor-infiltrating lymphocytes (TIL) and the induction of anti-tumor responses by IL-2 alone having proven to be promising approaches in cancer therapy. The present study investigated the cytotoxicity of LAK cells towards human leukemia cells. LAK cells were generated by culturing peripheral blood mononuclear cells from normal donors for six days in the presence of recombinant interleukin 2 (IL-2). Cytotoxicity was evaluated using a standard 4-h chromium-release assay. A significant lysis of fresh uncultured leukemia cells by IL-2-activated killer cells could be detected in 77 of 150 leukemias examined. The mean Cr-release was 35.7 +/- 12.9% in the LAK cell-sensitive vs 9.9 +/- 5.9% in the resistant leukemias. With a view to the therapeutic utilization of the LAK-cell system, we attempted to improve the efficiency of its cytotoxic mechanisms. Combined application of IL-2 and interferon-alpha, interferon-gamma, or tumor necrosis factor-alpha in cultures for generation of activated killer cells significantly improved the effectiveness of cytotoxic mechanisms. Our results suggest that the performance of adoptive immunotherapy with ex vivo-activated LAK cells and the in vivo induction of cytotoxic immune responses by IL-2 alone or combined with different lymphokines or cytokines may be of value in treating human leukemia, especially when the tumor burden is low, e.g. during maintenance therapy or after bone marrow transplantation to eliminate minimal residual disease or in early relapse.     

Biodistribution of lymphokine-activated killer (LAK) cells in Wag rats after hepatic-artery or jugular-vein infusion.

This study deals with the biodistribution of syngeneic radiolabeled lymphokine-activated killer (LAK) cells in Wag rats after infusion via the hepatic artery or the jugular vein. The biodistribution of 111Indium-labeled LAK cells was evaluated using serial whole-body gamma camera imaging. Furthermore, we investigated 2 factors that might influence the biodistribution of these effector cells: purity of LAK cells and administration of interleukin-2 (IL-2). After injection of 111Indium-labeled LAK cells via the hepatic artery or via the jugular vein we could detect important differences in the biodistribution pattern up to 5 hr after injection. LAK cells administered via the jugular vein were all found in the lungs up to 2 hr after injection and then redistributed to the liver and to the spleen. LAK cells administered via the hepatic artery were all found in the liver after injection and redistributed after 2 hr mainly to the spleen. About 8 hr after injection we could no longer detect any differences in the biodistribution pattern according to the route of administration. Biodistribution was followed for up to 72 hr after injection but the pattern showed no change after 8 hr, whichever the route of administration. A purified adherent-LAK population, a large granular lymphocyte culture with only 6% T cells, showed the same distribution pattern as standard LAK cells (40% T cells). Infusions of 40,000 units of IL-2 per day, starting 3 days before and continuing after administration of radio-labeled LAK cells, accelerated the redistribution of these cells by both routes of administration. We conclude that up to 2 hr after local infusion, a high concentration of LAK cells in the first capillary bed can be obtained. Therefore, local administration of LAK cells may be more effective against tumors.     

Expression and release of adhesion molecules by human acute myelogenous leukemia blasts

The expression and release of adhesion molecules by acute myelogenous leukemia (AML) blasts was investigated in vitro. For most patients AML blasts expressed relatively low levels of membrane-bound L-selectin and Intercellular adhesion molecule 1 (ICAM-1), but their soluble forms were detected in the supernatants for the majority of patients when AML blasts were cultured in vitro. These in vitro levels of SL-selectin and sICAM-1 were considerably lower than the normal serum levels. Divergent and relatively small alterations in SL-selectin and sICAM-1 levels were usually observed when exogenous growth factors were present during AML blast culture, whereas increased SL-selectin levels were observed after coculture of AML blasts and normal leucocytes. E- and P-selectin were neither expressed nor released by AML blasts. We conclude that AML blasts are a source of soluble adhesion molecules.     

Glycoproteic nature of surface molecules of effector cells with lymphokine-activated killer (LAK) activity. Evidence that T11, T8 or T3 molecules are not involved in tumor-cell lysis by LAK effector T cells

Human peripheral blood mononuclear cells cultured in the presence of interleukin-2 (IL-2) acquire the capability of lysing NK-resistant fresh tumor target cells. In an attempt to delineate the surface structure(s) present on the effector cells, the latter were first treated with different amounts of pronase and neuraminidase. The effect of the enzymes on cytolytic activity against fresh melanoma cells was evaluated and compared with the NK-like activity against K562 target cells of the same effector population. At a pronase concentration of 0.01 mg/ml, no inhibition of NK-like activity was detected, whereas LAK activity was inhibited by more than 75%. In addition, neuraminidase had no effect on NK-like activity, even at 1 U/ml, whereas as little as 0.03 U/ml inhibited LAK activity by more than 75%. Metabolic inhibition of N-linked glycosylation with Tunicamycin prevented the generation of LAK activity, even when added late (18 hr before termination of the culture). Tunicamycin, on the other hand, had no effect on the boost of NK activity induced by IL-2. Provided that LAK activity can also be generated in T-cell (E-rosetting) populations, in the presence of adherent cells, we analyzed the inhibitory activity of monoclonal antibodies (MAbs) to T11, T3 and T8 molecules. While all these MAbs strongly inhibited the specific target cell lysis by alloreactive CTLs, they had no effect on the LAK activity.     

Adoptive immunotherapy using lymphokine-activated killer (LAK) cells and interleukin-2 for recurrent malignant primary brain tumors

Summary Ten patients with recurrent malignant primary brain neoplasms were treated with adoptive immunotherapy using lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2). Nine patients had supratentorial glioma and they received multiple intratumoral instillations of LAK cells through reservoir-catheter system or burrhole. The other patients with disseminated subarachnoid metastases from posterior fossa medulloblastoma received immunotherapy via lumbar subarachnoid route. A partial and transient clinical response was observed in two patients following the therapy, and a cystic transformation of the essentially solid tumor was noted on the CT scans of these two patients. No significant clinical or radiological response to the treatment was observed in the remaining 8 patients. The results of this preliminary study reveal limitations of the regional intratumoral adoptive immunotherapy using currently available techniques and provide sufficient evidence of its effectiveness to warrant further investigations.     

Microvascular endothelial cells increase proliferation and inhibit apoptosis of native human acute myelogenous leukemia blasts

Interactions between acute myelogenous leukemia (AML) blasts and neighbouring endothelial cells in the bone marrow seem important both for disease development and susceptibility to chemotherapy. We investigated the effects of soluble mediators released by microvascular endothelial cells on native human AML cells. AML cells derived from 33 patients were cocultured with microvascular endothelial cells, separated by a semipermeable membrane. We investigated the effect of coculture on AML cell proliferation, viability/apoptosis and cytokine release. Coculture increased AML cell proliferation, and this growth enhancement included the clonogenic leukemia cell subset. Increased release of several soluble mediators was also detected (interleukin 3, interleukin 6, granulocyte-macrophage and granulocyte colony-stimulating factors) in cocultures. Our cytokine neutralization experiments suggest that an intercellular crosstalk involving several soluble mediators contribute to the increased leukemia cell proliferation. The presence of endothelial cells had an additional antiapoptotic effect on the AML cells. The endothelial cells did not have any growth-enhancing effect on native human acute lymphoblastic leukemia cells. Our in vitro results suggest that the release of soluble mediators by microvascular endothelial cells supports leukemic hematopoiesis through paracrine mechanisms by direct enhancement of AML blast proliferation and by inhibition of leukemic cell apoptosis.     

A case of pericarditis carcinomatosa showing good response following local transfer of lymphokine-activated killer (LAK) cells

A good therapeutic response following local transfer of lymphokine-activated killer (LAK) cells was obtained in a patient with cardiac tamponade due to breast carcinoma. A 41-year-old female was admitted with complaints of dyspnea and tachycardia. She had undergone left mastectomy at the age of 37 years and had received continuous oral administration of tamoxifen. Chest roentgenogram revealed cardiomegaly (CTR = 65%) and cardiac echogram showed marked retention of pericardial effusion. The cytology of the effusion was class V (adenocarcinoma). Cardiac tamponade proved refractory to combination chemotherapy using adriamycin, cyclophosphamide and 5-fluorouracil, and the effect of paracentesis was only temporary. Autologous peripheral blood lymphocytes were obtained through the cubital vein and cultured in vitro with 2 units/ml of human recombinant IL-2, (TGP-3, Takeda Pharm. Co.). After 4 days of cultivation, LAK cells were transferred intrapericardially 3 times. The cumulative infusion dose was 1.2 X 10(8) cells and the amount of combined IL-2 administration was 100 units/each transfer. Twenty-four days after initial infusion of LAK cells, the effusion disappeared. After then, recurrence has not been observed for 287 days. This case is the first trial of LAK therapy against pericarditis carcinomatosa and seems to be a useful way of treating this uncontrollable state without any serious side effects.     

Suppression of the generation of lymphokine-activated killer (LAK) cells by serum-free supernatants of in vitro maintained tumour cell lines

Serum-free supernatants from in vitro maintained gastrointestinal cancer and melanoma cell lines inhibit the generation of lymphokine (IL-2) activated killer (LAK) cells in a time and dose-related manner. Concentrations as low as 5% can inhibit the generation of LAK cytotoxicity but inhibition of proliferation is not observed until higher concentrations are included in the culture system. Inhibition is not observed with supernatants from a breast cancer cell line nor with supernatants from normal cells. There was complete concordance between the capacity of the tumour cells themselves to inhibit LAK generation and the presence of inhibitory activity in the corresponding supernatant. The inhibitory factor(s) is stable after heating to 44 and 56 degrees C. Production of the inhibitory factor(s) is sensitive to metabolic inhibitors and has a molecular weight greater than 25 kD. The inhibition of LAK cell stimulation by tumour cells may partially explain the failure of adoptively transferred LAK cells and IL-2 therapy to cause tumour regression in man.     

Increased susceptibility of IFN-gamma-treated neuroblastoma cells to lysis by lymphokine-activated killer cells: participation of ICAM-1 induction on target cells.

We have investigated the effect of interferon-gamma (IFN-gamma) treatment of neuroblastoma cells on the susceptibility to lysis by lymphokine-activated killer (LAK) cells and examined the participation of cell-adhesion molecules on the target cells in LAK cell lysis. Untreated neuroblastoma cells expressed lymphocyte-function-associated antigen 3 (LFA-3) and neural-cell-adhesion molecule (NCAM), but did not express MHC-class-I, MHC-class-II, or intercellular-adhesion molecule I (ICAM-I). IFN-gamma treatment of neuroblastoma cells induced the expression of MHC-class-I and ICAM-I antigens, but did not affect the expression of MHC-class-II, LFA-3, and NCAM. This was accompanied by an increased susceptibility to lysis by LAK cells. Anti-ICAM-I antibody inhibited partially the increased sensitivity of IFN-gamma-treated neuroblastoma cells to LAK cell lysis, and blocked completely the increase in binding of LAK cells observed after IFN-gamma treatment of the target cells. These results suggest that the increased LAK sensitivity of IFN-gamma-treated neuroblastoma cells is partially attributable to the induction of ICAM-I on neuroblastoma cells and indicate that post-binding events also play a role in the increased sensitivity to LAK cell lysis observed after IFN-gamma treatment.     

Induction of murine lymphokine-activated killer-like cells by Corynebacterium parvum (C. parvum) in vitro: lysis of tumor cells and macrophages by C. parvum-induced killer cells.

In vitro culture of murine spleen cells with Corynebacterium parvum (C. parvum) was found to induce lymphokine-activated killer (LAK)-like cells capable of killing both natural killer (NK)-sensitive and NK-resistant tumor cells as well as syngeneic macrophages (M phi). The induction of LAK-like activity by C. parvum was significantly inhibited by anti-interleukin-2 (IL-2) or anti-interferon (IFN) alpha, beta antibody (Ab), and it was further inhibited by the combination of two Abs, suggesting that the generation of killer cells by C. parvum was dependent on IL-2 and IFN(s) produced in the culture. It was considered that M phi were important in the induction of LAK-like cells by C. parvum because the depletion of M phi from spleen cells before culture with C. parvum significantly reduced the induction of killer activity. The majority of effectors mediating both tumor cells and M phi were Thyl+ and asialo-GM1 (aGM1)+, and the lysis of M phi by C. parvum-induced killer cells could be inhibited by the addition of cold YAC-1 tumor cells and P815 tumor cells, suggesting that the same population of effectors recognized tumor cells and M phi. These results demonstrated a possibility that the killing of M phi by C. parvum-induced killer cells might down-regulate anti-tumor effects of C. parvum.     

Generation and characterization of lymphokine-activated killer cells against fresh human leukemia cells

Lymphokine-activated killer (LAK) cells generated from 15 acute leukemia patients in remission showed significant levels of cytotoxicity against Daudi 1A4, a natural killer-resistant cell line. This indicates that lymphocytes of leukemia patients in remission could respond to interleukin-2 to generate conventional LAK cells. However, LAK cells caused lysis of autologous leukemia cells at considerably lower levels in seven out of the 15 patients, with the exception of one case (48.6% cytolysis). None of the remaining eight patients exhibited LAK activity against autologous leukemia cells. On the other hand, patients' LAK could lyse allogeneic leukemia cells including those resistant to autologous LAK. Thus, patients' LAK seem not to be defective in lysis of leukemia cells. In the cold target competition analysis, the binding of patients' LAK to leukemia cells could be inhibited by autologous and allogeneic leukemia cell competitors, implying that almost all leukemia cells could be recognized by patients' LAK. Most LAK cells from normal donors showed significant lysis of allogeneic leukemia cells, but some leukemia cells were found to be resistant to lysis. LAK cells against both leukemia cells and Daudi 1A4 were phenotypically heterogenous, and were predominantly observed in the T3- fraction in the precursor phase. In the effector phase, whereas LAK activity against leukemia cells was also predominantly shown in the T cell-depleted fraction, similar levels of LAK activity against Daudi 1A4 were found in both the T cell-depleted and -enriched fractions.