Molecular mechanisms of cytolysis induced by human lymphokine-activated killer cells

It has been shown that lysis of tumor target cells caused by lymphokine-activated killers is possible both upon a direct contact and in the presence of isolated nongranular cytotoxic proteins. The contact of cytolytic lymphocytes with K-562 cells leads to Fas L activation on the lymphocyte membrane and secretion of a broad spectrum of soluble cytotoxic proteins immunologically related to Tag 7 described earlier. These proteins can form inactive complexes, which are reactivated upon heating and addition of ATP. The proteins induced discrete cytolytic processes in tumor cells, differing in the rate of cytolysis and the mechanism of the apoptotic signal transduction. Fast processes (revealed in 3 h) mediated by caspases, and slow ones (in 24 h) with the supposed involvement of mitochondria were detected. A scheme for the lymphokine-activated killer interaction with target tumor cells is proposed.     

Immunomodulatory effects of systemic low-dose recombinant interleukin-2 and lymphokine-activated killer cells in humans

Summary The adoptive immunotherapy of human cancer using lymphokine-activated killer (LAK) cells in combination with high-dose systemic recombinant interleukin-2 (rIL-2) has been associated with global changes in several hematological and immunological parameters while imposing profound toxicity on patients. We have evaluated an alternative LAK cell therapy utilizing low-dose systemic rIL-2 in 27 consecutive patients with metastatic cancer. We report that the administration of systemic low-dose rIL-2 is also characterized by significant changes in immunological and hematological parameters, which are qualitatively similar to those induced by high-dose rIL-2. Low-dose systemic rIL-2, given by i.v. bolus, is cleared to baseline levels within 240 min of administration. The induction of lymphocytosis and eosinophilia, which has characterized other protocols, is also a feature of this protocol. In addition, low-dose systemic rIL-2/LAK cell immunotherapy results in increased peripheral blood mononuclear cell (PBMC) expression of T-cell activation markers such as OKIa, OKT10 and IL-2 receptor. PBMC sampled approximately 100 h after the final infusion of LAK cells demonstrated a statistically significant increase in their ability to kill natural killer (NK)-sensitive and NK-resistent cell lines such as K562 and Daudi compared to baseline values (P <.05). These data suggest that rIL-2-based immunotherapy using low-dose rIL-2 is capable of inducing quantitative hematological and immunological changes while (in combination with LAK cells) retaining the ability to mediate tumor regressionin vivo. Dr. Eberlein was a recipient of an American Cancer Society Career Development Award. This work is supported in part by NIH Grant CA-40555 and the Clinical Research Center Grant 20-9299     

Generation of lymphokine-activated killer cell activity by low-dose recombinant interleukin-2 and tumor cells

The generation of lymphokine-activated killer (LAK) cells in vitro has been reported to require 100-1000 units of recombinant interleukin-2 (IL2). In this study we investigated the generation of human LAK cells with low-dose IL2 (1-10 U) in combination with human tumor cell lines. A significant LAK activity was generated within 3- to 5-days culture of PBL. Among six human tumor cell lines tested, the K562 cell line had the greatest stimulating activity, and the degree of cytotoxicity was comparative to that of PBL stimulated with higher doses of IL2 alone. The origin of this LAK activity was primarily the E(-) rosetting cell population. Cocultures of E- cells with 1 U/ml IL2 plus K562 had significantly higher cytotoxicity (P less than 0.05) compared to using E+ cells. Phenotypic analysis indicated that 1 U/ml IL2 plus K562 cell stimulation enhanced CD56+ and CD16+ cells. These studies suggest that very low dosages of IL2 with stimulator tumor cells can generate LAK activity comparable to that generated with high dosages of IL2 alone.     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Induction of lymphokine-activated killer cells from rat thymocytes using recombinant human interleukin-2

Summary Using a 4-h ⁵¹Cr release assay, we observed that thymocytes from Fischer strain rats incubated with recombinant human interleukin-2 (rhIL-2) developed cytotoxicity to YAC-1 lymphoma, 9L-glioma, and B-16 melanoma cells (effector/target ratio =25/1). Induction of the lymphokine-activated killer (LAK) cells was as follows: (1) when 5×10⁶/ml thymocytes were cultured with various concentrations of rhIL-2 (50, 125, 250, 500, or 1,000 units/ml) for 4 days, no cell proliferation was observed at any concentration. However, the LAK cells showed significant cytotoxicity toward all tumor cells at more than 50 units/ml. (2) When 5×10⁶/ml thymocytes were cultured for 1 to 6 days with 250 units/ml of rhIL-2, the harvested cell count decreased markedly after the 2nd day. The cytotoxicity of all the tumor cells became significant after the 2nd day, with peak activity on the 4th day. In rat splenocytes, on the other hand, the LAK cells could not be identified because rat splenocytes developed nonspecific cytotoxicity in medium containing fetal calf serum without adding rhIL-2.     

Interleukin-6 does not support interleukin-2 induced generation of human lymphokine-activated killer cells

Summary The activity of lymphokine-activated killer (LAK) cells is supported by various cytokines. The objective of this study was to see if recombinant interleukin-6 (IL-6) either alone or in combination with interleukin-2 (IL-2) has any effect on the generation of LAK cells. Peripheral blood mononuclear cells of healthy donors were cultured for 4 or 6 days with both cytokines either alone or in combination. LAK activity against K562 and natural killer-resistant Daudi cells was assessed by a 4-h and an 18-h⁵¹Cr-release assay at various effector to target ratios. IL-6 alone in increasing concentrations did not induce LAK cell activity. Neither additive nor synergistic effects of IL-6 with IL-2 were observed. Immunofluorescence analysis with phycoerythrin-conjugated anti-CD56 antibody demonstrated that IL-6 could not maintain or increase the number of CD56-positive cells over a 6-day culture period. These results suggest that IL-6 does not support LAK cell generation by itself or increase LAK cell activity in combination with IL-2.     

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA

Summary Lymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10–1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 µg/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10–30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [³H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were dependent on rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.     

Induction of lymphokine-activated killer cell against human leukemia cells in vitro

The induction of lymphokine-activated killer (LAK) cells against fresh human leukemia cells was investigated. Two thirds of the 62 leukemias examined were susceptible to the lytic effect of allogeneic IL-2 induced LAK cells in vitro. No substantial differences could be detected between myeloid or lymphoid leukemias or with regard to the FAB subtype or the immunophenotype. Culturing mononuclear cells from peripheral blood or bone marrow of leukemia patients with IL-2 resulted in an expansion of residual large granular lymphocytes and development of cytotoxic activity. The combination of IL-2 with IFN-gamma or the presence of tumor cells during the activation process led to an enhancement of LAK cell cytotoxicity. These results suggest that LAK cells may be useful in the treatment of leukemia.     

In vivo generation of lymphokine-activated killer cells by sensitization with interleukin 2-producing syngeneic T-lymphoma cells

Culture of C57BL/6 mouse spleen cells with syngeneic EL4 lymphoma cells resulted in no induction of killer cells reactive against EL4 cells. However, in vitro sensitization of C57BL/6 mouse spleen cells with interleukin 2 (IL-2)-producing EL4 lymphoma cells caused the generation of lymphokine-activated killer (LAK) cells, which lyse a variety of tumor cells. Consistent with an in vitro system, we demonstrate that Thy 1.2+, Ly2+, asialo GM1+ LAK cells were successfully induced by in vivo immunization with syngeneic IL-2-producing EL4 lymphoma cells.     

Anti-tumor efficacy of interleukin-2-activated killer cells in human neuroblastoma ex vivo

We studied the ex vivo sensitivity of continuously cultured neuroblastoma cells from 3 different patients towards interleukin-2-induced cell-mediated cytotoxicity. A mean (+/- SD) target cell lysis (4 h 51Cr release) of 49 +/- 11, 46 +/- 8, and 32 +/- 11% in SMS-SAN, LA-N-1, and SK-N-BE2 cell lines, respectively, was achieved when neuroblastoma cells were co-cultured at an effector-to-target (E:T) ratio of 50:1 with peripheral blood mononuclear cells (PBMC) that had been preincubated for 4 days in the presence of recombinant interleukin-2 (rIL-2; 100 U/ml). Under identical conditions, 93 +/- 9% of Daudi cells (a standard target for rIL-2-activated killer cells) were lysed. Preincubation of rIL-2-induced PBMC cultures in the presence of irradiated neuroblastoma targets (LA-N-1, SK-N-BE2) resulted in a significant cytolytic augmentation. At E:T ratios of 50:1 and 10:1, day-4 rIL-2/LA-N-1-stimulated PBMC produced 69 +/- 7 and 41 +/- 11% lysis of LA-N-1 cells, as compared to 46 +/- 8 and 22 +/- (mean +/- SD) 7% lysis by untargeted PBMC that were preincubated with rIL-2 (100 U/ml) in the absence of LA-N-1 target cells (p less than 0.05). Co-incubation of rIL-2-induced PBMC preparations with irradiated LA-N-1 and SK-N-BE2 cells, respectively, did not significantly enhance the cytolytic activity against other neuroblastoma targets and the standard Daudi cell line (p greater than or equal to 0.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

The augmentation of lymphokine-activated killer activity following pulsing of human peripheral blood mononuclear cells with recombinant human interleukin-2

Summary The short-term exposure of peripheral blood mononuclear cells (PBMC) to recombinant human interleukin-2 (rhIL-2) at 37°C leads to the generation of lymphokine-activated killer (LAK) activity similar in magnitude to that obtained by the exposure of PBMC to rhIL-2 continuously for 3–5 days. In order to investigate whether the required signal for LAK induction occurred during the short exposure to rhIL-2 or at a later point in the induction phase, PBMC were exposed to rhIL-2 for 1 h at 4°C and then exposed to a low-pH wash to remove bound IL-2 from its receptor. PBMC treated in such a way showed increased LAK activity and proliferation compared to cells exposed to rhIL-2 alone. Expression of the p55 () subunit of the IL-2 receptor was also increased. In order to cause the augmentation, a lowering of the pH below 4.0 was necessary, and exposure of PBMC to low pH alone (in the absence of rhIL-2) failed to cause activation. Another relevant feature was a transient increase in the expression of the p75 subunit of the IL-2 receptor ( chain) immediately following the exposure to low pH and the release of interferon , tumour necrosis factor and IL-6; activation was blocked by the inclusion of neutralising antisera raised against rhIL-2 and interferon , thus demonstrating that the endogenous release of these cytokines is important for activation.     

The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo

We showed previously that adoptive immunotherapy with the combination of LAK cells and recombinant IL 2 (RIL 2) can markedly reduce pulmonary micrometastases from multiple sarcomas established 3 days after the i.v. injection of syngeneic tumor cells in C57BL/6 mice. In this report, we analyzed the factors required for successful therapy. Titration analysis in vivo revealed an inverse relationship between the number of pulmonary metastases remaining after treatment and both the number of LAK cells and the amount of RIL 2 administered. Fresh or unstimulated splenocytes had no anti-tumor effect; a 2- to 3-day incubation of splenocytes in RIL 2 was required. LAK cells generated from allogeneic DBA (H-2d) splenocytes were as effective in vivo as syngeneic, C57BL/6 (H-2b) LAK cells. The anti-metastatic capacity of LAK cells was significantly reduced or eliminated when irradiated with 3000 rad before adoptive transfer. The combined therapy of LAK cells plus RIL 2 was shown to be highly effective in mice immunosuppressed by 500 rad total body irradiation and in treating macrometastases established in the lung 10 days after the i.v. injection of sarcoma cells. Further, reduction of both micrometastases and macrometastases could also be achieved by RIL 2 alone when administered at higher levels than were required with LAK cells. The value of LAK cell transfer and of IL 2 administration for the treatment of tumors established at other sites is currently under investigation.     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector(R) electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/106 cells (range of 107.6-1079.3 pg /106 cells/24 h) compared to mock transfected CIK cells (31 pg/106 cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 +/- 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 +/- 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line.     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies

Clinical effects and side effects were studied in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of normal donors by means of initial stimulation with pokeweed mitogen (PWM). Six of 15 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastasis, pleural effusion and primary tumor. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Generally toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently except one patient who showed preshock like symptom accompanied with leukocytopenia and continuous hypotension immediately after infusion but was finally rescued. In the patients who received more than 10¹¹ of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies appeared in the patients. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells.Abbreviations PWM pokeweed mitogen - LAK lymphokine-activated killer - IL-2 interleukin 2 - PEL peripheral blood lymphocytes - TIL tumor-infiltrating lymphocytes - GVHD graft-versus-host disease - HLA human leukocyte antigen     

In vitro and in vivo suppression of interleukin-2-activated killer cell activity by chimeric proteins between interleukin-2 and Pseudomonas exotoxin.

The biological effects of IL-2 are mediated through high (complex of alpha and beta chain) or intermediate (beta chain) affinity IL-2 receptors. Previously, chimeric proteins composed of IL-2 and Pseudomonas exotoxin (IL-2-PE) were shown to be specifically cytotoxic to cells bearing IL-2 receptors. It has also been shown that IL-2-PE chimeric proteins can abrogate T cell-mediated immune response in vitro. In the current study, we have investigated the effects of IL-2-PE on LAK activity both in vivo and in vitro. We administered either IL-2-PE40 (comprised of IL-2 and 40-kDa portion of PE) or IL-2-PE66 (comprised of IL-2 and 66-kDa molecule of PE) to normal C57BL/6 mice for 3 or 8 days and LAK activity was assessed in various organs of mice. We found that IL-2-PE40 generated LAK activity in various compartments of mice and the level of activity was slightly lower than that observed with an equivalent amount of recombinant (r) IL-2 alone. However, IL-2-PE66 failed to generate LAK activity which would have been induced due to an equivalent concentration of rIL-2. IL-2-PE66 also did not induce LAK activity from the splenocytes during in vitro culture while IL-2-PE40 generated good LAK activity. An equivalent amount of IL-2 also generated potent LAK activity. The suppression of LAK activity by IL-2-PE66 was also evident in cells preactivated with IL-2; however, this inhibition was partial. The suppressive activity of IL-2-PE66 was shown to be mediated through IL-2 receptor interactions as excess amounts of rIL-2 were able to abrogate its effect. Both IL-2 toxins were equivalently cytotoxic to IL-2 receptor-bearing HUT 102 cells and both were able to compete from high and intermediate affinity IL-2 receptors. Taken together, our data indicate that IL-2-PE66 is highly cytotoxic to LAK cells while IL-2-PE40 is less cytotoxic. Thus, data from our study and from other published reports indicate that IL-2-PE66 is more potent immunosuppressive agent than IL-2-PE40.     

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: II. characterization of cells cultured ex vivo and their contribution to the in vivo immunomodulation

Summary Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines (LCL). We studied three groups of BL lines for their capacity to interact with allogeneic lymphocytes. The results showed that as long as the lines have the group I phenotype, they do not stimulate allogeneic T lymphocytes irrespective whether they carry the EBV genome. The group II and III cells are stimulatory. Generally there was no correlation between sensitivity to lymphocyte-mediated lysis and the phenotype of the lines. In one set of lines, the group I cells had higher sensitivity to both natural killer and lymphokine-activated killer effectors compared to the group II or III lines. However, such correlation could not be seen with the other two sets of lines. Among the phenotypic features investigated, expression of the adhesion molecules LFA-1 and LFA-3 correlated with the tendency for cell aggregation.     

In vivo migration and tissue localization of highly purified lymphokine-activated killer cells (A-LAK cells) in tumor-bearing rats

Our laboratory has previously reported that the adoptive transfer of highly purified lymphokine-activated killer cells (adherent-LAK, A-LAK) into Fischer 344 (F344) rats bearing established lung or liver micrometastases effectively reduced the resultant tumor growth more than 90%, leading to significant increases in animal survival (Cancer Res. 49, 1441, 1989). To begin to investigate the mechanism(s) by which A-LAK cells mediate this anti-tumor effect, we studied their migration patterns in F344 rats bearing experimentally induced lung and liver metastases as well as subcutaneous tumors. A-LAK cells which were phenotypically 95 to 100% natural killer cells/large granular lymphocytes were labeled with either 51Chromium or fluorescein diacetate (so as to be visualized microscopically). Intravenous injection of such labeled A-LAK cells did not show significant differences in their tissue distribution patterns in tumor-bearing versus normal rats, even when high levels of exogenous recombinant interleukin-2 (rIL-2) was administered. A-LAK cells first migrated to the lungs and then subsequently migrated to the liver and spleen as early as 2 to 6 hr following iv injection. The kinetics of exit of A-LAK cells from the pulmonary capillary beds was not significantly different in rats bearing 3-day micrometastases or 14-day macrometastases compared to normal rats. Moreover, the presence of metastases in the liver did not alter the extent or kinetics of entry of A-LAK cells into the liver even in the presence of exogenously administered rIL-2. Finally, in rats bearing subcutaneous tumors, no evidence could be obtained that A-LAK cells were selectively localized to the tumor site. Tissue sections of livers from metastases-bearing animals injected with fluorescein diacetate labeled A-LAK cells did not demonstrate significant numbers of A-LAK cells infiltrating tumor nests with or without the administration of exogenous IL-2. These data suggest that A-LAK cells may mediate tumor regression in vivo by direct and indirect mechanisms, possibly through the secretion of cytokines and/or the recruitment of secondary effector cells.     

Use of human leukocyte antigen-mismatched allogeneic lymphokine-activated killer cells and interleukin-2 in the adoptive immunotherapy of patients with malignancies.

Clinical effects and side effects were investigated in the adoptive immunotherapy of patients bearing malignant diseases using human leukocyte antigen (HLA)-mismatched allogeneic lymphokine-activated killer (LAK) cells. Allogeneic LAK cells were induced from peripheral blood lymphocytes (PBL) of healthy donors with the same blood types as those of patients. Recently we succeeded in increasing the proliferation rate and enhancing the cytotoxic activity of LAK cells by means of initial stimulation with pokeweed mitogen (PWM, PWM-LAK cells). Five of 12 patients applied in the adoptive immunotherapy showed clinical effects such as partial or complete regression of pulmonary metastases and pleural effusion. All pulmonary metastatic lesions were eliminated in one case by this adoptive immunotherapy combined with chemotherapy. Toxic effects were chillness, fever and general fatigue which were reversible, and no allergic side effects occurred even though allogeneic LAK cells were injected frequently. In the patients who received more than 10(11) of allogeneic LAK cells, anti-HLA class I antibodies appeared without any evidence of autoantibody. However, immunological side effects were never experienced after injection of allogeneic LAK cells even when the anti-HLA class I antibodies existed in the patients; this phenomenon suggests the safety of the adoptive immunotherapy using allogeneic LAK cells. Taken together, allogeneic LAK cells could be considered as alternative therapy for patients with malignancies who could not supply sufficient materials of autologous LAK cells. Recently, LAK cells, particularly PWM-LAK cells were found to obtain significantly potent and prompt lectin-dependent cell-mediated cytotoxicity (LDCC). All tumor cells confluent in microtest plate well could be annihilated by PWM-LAK cells plus PWM less than 8 hours. New immunotherapy using PWM-LAK cells or lectin-stimulated LAK cells with PWM or other lectins is discussed.     

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

Summary The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10–200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of TNF suggesting interferon-independent activation. In further experiments after i.p. injection of TNF peritoneal exudate cells (PECs) only killed YAC-1 targets in a 4-h assay. There was no additional killing in an 18-h assay towards neither YAC-1 cells or P815 cells, suggesting that macrophages were not involved. Furthermore TNF was also active in vitro by activating NK cells in isolated human peripheral blood cells. However in the PECs stimulated in vitro no significant induction of cytotoxic capacities by TNF was measured. Our data suggest that the action of TNF is not restricted to the lysis of tumor cells but can also induce immunological properties in the host defense against virus infections and neoplasms.