Induce Lymphokine-activated Killer Cells Research Articles

Staccato pulse interleukin-2 (IL-2) in metastatic melanoma (MM) previously treated with checkpoint inhibitors (CPI).

e21022 Background: The CPI ipilimumab, nivolumab, and pembrolizumab have been adopted into common use for patients (pts) with MM. While their clinical activity is undeniable, a substantial fraction of pts treated with these agents experience disease progression. Additional approaches are therefore needed. Intravenous IL-2 given via daily single intravenous infusions (pulses) has been developed to mitigate toxicity while maintaining anticancer activity against MM. Staccato pulse IL-2 is based on three prior observations. Daily IL-2 schedules have previously been demonstrated to induce Lymphokine Activated Killer cell (LAK) activity [Mitchell, 1989]. LAK generated by IL-2 then subsequently exposed to more IL-2 display enhanced cytotoxicity in vitro [Hank, 1990]. Increased numbers of LAK are seen in pts with melanoma treated with daily IL-2 [Quan, 2011]. Methods: In this retrospective study, ten pts with MM were treated with IL-2 18 Million IU/M2 intravenously over 15-30 minutes on days 1-3 and 21.6 Million IU/M2 intravenously over 15-30 minutes on day 5 on an outpatient basis. Cycles were repeated every 3 weeks. Results: Characteristics: 8 females/ 2 males, median age-56 (range: 21-74), median ECOG-1 (0-1); common disease sites: lymph nodes (12), subcutaneous (6), lungs (5), soft tissue (4). Prior CPI: Ipilimumab (8); Pembrolizumab (5); Nivolumab (2). Common toxicities: nausea/emesis (8), myalgia/arthralgia (5), sinus/catarrhal symptoms (5), fatigue (5), and hypotension requiring intravenous fluids (5). No pts required hospitalization for toxicity related to therapy. Median number of cycles: 3 (2-8). Four pts have had partial responses (total response rate = 40%; 95% CI: 10-70%). The median duration of response exceeds 7.4+ months. One pt having had resolution of prior lung, lymph node, and peritoneal metastases (partial response of 24.5+ months) underwent surgical resection of the residual intraabdominal disease focus and is free of disease at 36.5+ months. Overall, responses have occurred in lung, lymph nodes, subcutaneous, bones, peritoneum, small bowel, and soft tissue sites. Conclusions: Staccato pulse intravenous IL-2 has activity in melanoma pts previously treated with CPI.

Activation of CD38 by Interleukin-8 Signaling Regulates Intracellular Ca2+ Level and Motility of Lymphokine-activated Killer Cells

CD38 is an ADP-ribosyl cyclase, producing a potent Ca(2+) mobilizer cyclic ADP-ribose (cADPR). In this study, we have investigated a role of CD38 and its regulation through interleukin-8 (IL8) signaling in lymphokine-activated killer (LAK) cells. Incubation of LAK cells with IL8 resulted in an increase of cellular cADPR level and a rapid rise of intracellular Ca(2+) concentration ([Ca(2+)](i)), which was sustained for a long period of time (>10 min). Preincubation of an antagonistic cADPR analog, 8-Br-cADPR (8-bromo-cyclic adenosine diphosphate ribose), abolished the sustained Ca(2+) signal only but not the initial Ca(2+) rise. An inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist blocked both Ca(2+) signals. Interestingly, the sustained Ca(2+) rise was not observed in the absence of extracellular Ca(2+). Functional CD38-null (CD38(-)) LAK cells showed the initial rapid increase of [Ca(2+)](i) but not the sustained Ca(2+) rise in response to IL8 treatment. An increase of cellular cADPR level by cGMP analog, 8-pCPT-cGMP (8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate), but not cAMP analog or phorbol 12-myristate 13-acetate was observed. IL8 treatment resulted in the increase of cGMP level that was inhibited by the IP(3) receptor blocker but not a protein kinase C inhibitor. cGMP-mediated Ca(2+) rise was blocked by 8-Br-cADPR. In addition, IL8-mediated LAK cell migration was inhibited by 8-Br-cADPR and a protein kinase G inhibitor. Consistent with these observations, IL8-induced migration of CD38(-) LAK cells was not observed. However, direct application of cADPR or 8-pCPT-cGMP stimulated migration of CD38(-) cells. These results demonstrate that CD38 is stimulated by sequential activation of IL8 receptor, IP(3)-mediated Ca(2+) rise, and cGMP/protein kinase G and that CD38 plays an essential role in IL8-induced migration of LAK cells.

Immunomodulatory activity of resveratrol: discrepant in vitro and in vivo immunological effects

trans-Resveratrol is a dietary polyphenolic compound present in grapes, which has been shown to exhibit strong anti-inflammatory, antioxidant, and chemopreventive activities. In this study we have compared the in vitro and in vivo effects of resveratrol on the development of various cell-mediated immune responses, including mitogen/antigen-induced T cell proliferation, induction of cytotoxic T lymphocytes (CTLs), interleukin-2 (IL-2) induced lymphokine activated killer cells, and cytokine production. We found significant suppression (>90%) of the mitogen/antigen-induced T cell proliferation and development of allo-antigen specific CTLs in vitro with resveratrol at a concentration of 25 μM. Intragastric administration of resveratrol (2 mg daily) to mice for 4 weeks showed no effect on age-related gain in body weight, peripheral blood cell counts (WBC, RBC, or platelets), or the cellularity of bone marrow or spleen. The CD4 + and CD8 + T cells in spleen or colony-forming units-total in the marrow also remained unaffected by treatment with resveratrol. Spleen cells, which were stimulated in vitro after being removed from mice which had been administered resveratrol for 2 or 4 weeks, showed no significant change in IL-2 or concanavalin A induced proliferation of T cells or production of IL-2 induced lymphokine activated killer cells. Further, the production of in interferon-gamma and IL-12 was not affected by administration of resveratrol, but production of tumor necrosis factor-alpha was reduced. Even when conducted entirely in vivo, treatment with resveratrol was found to only marginally reduce allo-antigen induced T cell proliferation and the generation of CTLs in the draining lymph nodes. Thus, even though resveratrol strongly inhibits T cell proliferation and production of cytolytic cells in vitro, oral administration of resveratrol for 4 weeks does not induce hematologic or hematopoietic toxicity, and only marginally reduces the T cell-mediated immune responses.

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.

Lymphokine activated killer cells in murine coxsackievirus B3 myocarditis.

The purpose of the present study was to determine whether lymphokine activated killer (LAK) cells were involved in the development of coxsackievirus B3 (CB3) myocarditis in both the acute viremic (Experiment I) and the subacute aviremic (Experiment II) stages. To induce LAK cells, recombinant human interleukin-2 (IL-2) was administered to CB3-infected mice subcutaneously daily, starting on day 0 in Experiment I and on day 7 in Experiment II for 7 days, respectively. The treated groups were compared to infected controls. Splenic lymphocytes of IL-2 treated mice were further cultured in vitro in IL-2 containing medium for 7 days, and LAK cell activity, i.e., cytotoxic activity of the lymphocytes against EL-4 tumor cells and against cultured fetal myocytes, was assayed by 51Cr-release method. In Experiment I, histologic scores, myocardial virus titers, and LAK cell activity did not differ significantly between IL-2 treated and untreated groups. In contrast, in Experiment II, there were more cellular infiltration associated with severe necrosis and higher LAK cell activity against EL-4 cells and cultured myocytes in IL-2 treated than in untreated groups. The presence of LAK cells was demonstrated in the subacute stage of murine CB3 myocarditis. Thus, the behavior of LAK cell activity may vary with the course of myocarditis, and enhanced LAK cell activity may be involved in the development of the disease.

Combined effect of adoptive immunotherapy (AIT) with lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) and chemotherapy in tumor-bearing mice

We investigated beneficial effects of AIT with anticancer agents on survival of subcutaneous tumor-bearing mice and suppression of artificial lung metastasis, and optimal schedule of administration of each treatment in vivo. 7-8 weeks old C 57 BL/6 mice were inoculated s.c. with 5 x 10(6) B 16 melanoma cells, or i.v. with 2 x 10(5) B 16-F 10 melanoma cells. Mouse splenocytes were cultured with 3.5 x 10(3) JRU/ml interleukin 2 for 14 days, and induced LAK cells were harvested. Anticancer agents (Cx), CDDP or MMC were given i.p. in mice. 1 x 10(7) or 5 x 10(7) LAK cells were given either s.c. around the tumor or i.v. respectively, and 1.4 x 10(5) JRU/mouse IL-2 was administered s.c. for 6 days after LAK cell injection. Therapy groups were as follows #1: Cx day 3, AIT day 3-8. #2: Cx day 3, AIT day 6-11, #3: Cx day 8, AIT day 3-8. In therapy groups #1 and #2, we observed additive effects of AIT and anti-cancer agents in life-prolongation and suppression of lung metastasis. It was also shown that LAK induction in vivo was augmented by anticancer agents in groups #1 and #2, which might represent one of the mechanisms behind observed additive effects. Furthermore, our results suggest that therapeutic effects of the combination of AIT and anticancer agents depend on the schedule of administration.

Generation of Lymphokine-Activated Killer Activity in Rodents but Not in Humans Is Nitric Oxide Dependent

The role of nitric oxide (NO) in the generation of lymphokine-activated killer (LAK) cells was investigated. Here we report that L-arginine analog NG -monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide synthase, prevents LAK cell generation from cultured rat splenic cells. Accumulated NO endproduct nitrite (NO-2), as measured in the supernatants of rat splenic cells, correlated well with the generation of LAK cells. In contrast, cell proliferation induced by rIL-2 or by Con A was not affected by NMMA. Similarly, phenotypic expression of CD25 in rIL-2-stimulated cultures was unaffected. Furthermore, we could not observe differences in percentages of CD5-CD8+ cells (NK and LAK cell phenotype markers in rats) between rIL-2-stimulated cultures performed in the presence or absence of NMMA. LAK cell generation could no longer be blocked if NMMA was added to the rat cell cultures 24 hr after rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-arginine. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell generation. Finally, addition of sodium nitroprusside (SNP) which releases NO in cultures was able to overcome blocking effects of NMMA. To attempt the identification of NO-producing cells, lysosomotropic agent, L-leucine methyl ester (LME), was used. Generation of LAK cell activity was virtually abolished in cell cultures treated with LME. Addition of SNP to cultures, however, sufficed to restore LAK cell generation. These results suggest that LAK cell precursors depend on a exogenous NO supply from other cell types in order to display their full cytotoxic potential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did not affect generation of LAK cells from human peripheral blood or spleen mononuclear cells.

Immunological parameters in peripheral blood of patients with renal cell carcinoma before and after nephrectomy

To determine the effects of nephrectomy on the immune response of patients with renal cell carcinoma (RCC). Five patients with RCC were monitored before and over a period of up to 3 months after nephrectomy. The aspects measured were the phenotypic expression of markers on circulating lymphocytes, circulating concentrations of cytokines, markers of inflammatory and immune responses, and natural killer (NK) cell and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells (PBMC). The suppressive activity of patients' plasma on NK activity and ability to generate interleukin-2 (IL-2) induced LAK cells in PBMC of normal volunteers was also investigated. The results indicated that high CD4/8 ratios were present pre-nephrectomy with evidence of inflammatory responses and immune activation in some patients, particularly those with metastatic disease. The effect of nephrectomy was to decrease the inflammatory response and increase immune activation. Various defects in NK cell activity and LAK cell generation were demonstrated pre-surgery which slowly improved once the primary tumour had been removed and it is suggested that such defects could have contributed to tumour growth and development due to an ineffective immune response.

Local lnterleukin-2 Therapy for Cancer, and Its Effector Induction Mechanisms

This paper reviews the effectiveness of local interleukin-2 (IL-2) therapy and its effector induction mechanisms in vivo. Local therapy with IL-2 and/or lymphokine-activated killer (LAK) cells is associated with far fewer side effects than systemic treatment. Local infusions of IL-2 into malignant pleural effusions and the peritoneal cavity induce LAK cells and secondary production of other cytokines responsible for up- or down-regulation of LAK activity. An understanding of the regulatory mechanism of local LAK induction in vivo may provide the rationale for a more effective therapeutic modality for cancer in humans.

Experimental study on interleukin 6 activity against tumor; Regulation of interleukin-2 induced lak cell activity

The ability of human recombinant interleukin-6 (IL-6) to regulate the induction and cytotoxic function of lymphokine activated killer (LAK) cells from human fetal spleen was studied. The results showed that IL-6 alone was unably to induce fetal splenic mononuclear cells (FSMC) to develop functional LAK cells, nor was it able to affect the number of IL- 2- induced LAK cells and to alter the lytic activity of LAK cells against tumor cells in effector phase. However, when IL- 6 was used in combination with IL- 2 during the induction phase, the resulting LAK cells displayed considerably greater lytic, activity than that with IL-2 alone (P<0.01), and this effect was IL-6 dose-dependent. The possible machanism of the synergistic effect of IL-2 and IL-6 in LAK cell induction from human fetal spleen is discussed.

T-Cell Subsets Required for Intravesical BCG Immunotherapy for Bladder Cancer

Intravesical bacille Calmette-Guérin (BCG) has been shown in prospective randomized clinical trials to be the treatment of choice for superficial bladder cancer. In this investigation we evaluated the role of CD4 and CD8 lymphocytes in the antitumor response. Monoclonal antibodies to thy 1.2, CD8, CD4 and an isotype control were injected intravenously to deplete T cell populations. After depletion (verified by flow cytometry), BCG therapy was initiated. The results demonstrate that the depletion of either CD4 or CD8 T cell subsets eliminated BCG-mediated antitumor activity. Footpad delayed type hypersensitivity (DTH) was aborted only in CD4 depleted mice; it was essentially unchanged in CD8 depleted mice. However, the presence of DTH was not sufficient for induction of BCG-mediated antitumor activity. Exogenous IL-2 at levels sufficient to induce lymphokine activated killer cell activity did not substitute for CD4 cells. There was no evidence for the induction of protective immunity to the tumor after BCG therapy. These results demonstrate the requirement for T lymphocytes in BCG-mediated antitumor activity and further demonstrate that the presence of both CD4 and CD8 subsets are required. CD8 depletion experiments suggest that the presence of CD4-mediated DTH is not sufficient for the induction of antitumor activity. Furthermore, these data suggest that BCG-mediated antitumor activity is a localized phenomenon that does not induce protective immunity.

The characterization of peritoneal and pleural exudate cells from malignant effusions

The immune function of peripheral blood cells and cells from the pleural and abdominal effusions of patients with advanced cancer was compared to that of peripheral blood cells from controls. The parameters examined included lymphocyte subsets, natural killer (NK) cell activity, and anti-Daudi and lymphokine-activated killer (LAK) cell activity. The percentage of CD4+ pleural and peritoneal exudate cells (PEC) was significantly higher than the percentage of peripheral blood mononuclear cells (PBMC) in the patients. The percentage of CD8+CD11+ PEC and PBMC, being the suppressor T-cells, of the patients was increased compared with controls, while the percentage of CD8+CD11- PEC, being the cytotoxic T-cells, was identical to the PBMC of both patients and controls. The NK activity of PEC was significantly lower than that of PBMC in both patients and controls, and there was no correlation between the NK activity of PBMC and PEC. Although the anti-Daudi activity of PEC was markedly low, LAK cells with high activity could be induced by culture with interleukin-2 for 4 days. These results suggest that the immune function of cells in malignant effusions may be depressed due to a low population of cytotoxic T cells, low NK activity and increased suppressor T cells, while the local administration of interleukin-2 may induce LAK cells. Therefore, effective local immunotherapy for malignant effusions should not only augment effector cells, but also inhibit suppressor cells.

Induction of lymphokine-activated killer cells from nude mouse spleen cells by interleukin-4.

The induction of lymphokine-activated killer (LAK) cells from natural killer (NK) lineage cells by interleukin-4 (IL-4) was studied in vitro. Activation of nude mouse spleen cells by IL-4 generated cytotoxic cells, capable of killing NK-sensitive as well as NK-resistant tumor cells. The induction of peak lytic activity was demonstrated after 3 days of culture with IL-4. Surface marker analysis indicated that the majority of precursor cells were aGM1+, Thy1-, and the majority of effector cells were aGM1+, Thy1+, suggesting that IL-4 induced LAK cells from nude mouse spleen cells were similar to those from normal mouse spleen cells. The induction of nude mouse LAK cells by IL-4 was partially inhibited by anti-IL-4 or anti-interferon (IFN)-alpha,beta antibody, and it was further inhibited by the combination of two antibodies, suggesting that IFN-alpha,beta production was associated with LAK induction of NK lineage cells by IL-4.

Lymphokine-activated killer cell regulation of T-cell-mediated immunity to Candida albicans.

Monocytes are important accessory cells in the activation of T cells for specific antigen recognition yet little is known of their regulation. We demonstrated here that interleukin-2 (IL-2)-induced human lymphokine-activated killer (LAK) cells can inhibit monocyte antigen presentation, depending on the state of differentiation of the monocytes. Adherent monocytes cultured for 4 days in medium or granulocyte-macrophage colony-stimulating factor (GM-CSF) were found to equally process and present intact Candida albicans to autologous Percoll gradient-isolated T cells, as measured by [3H]thymidine uptake. However, only the GM-CSF-cultured monocytes were functionally inhibited by autologous 4-day IL-2-induced LAK cells. Even soluble candidal cell wall mannoprotein antigens could not be presented by these monocytes after exposure to LAK cells. Pretreatment of these monocytes with LAK cells for 1 h, followed by subsequent removal of the nonadherent LAK cells, was sufficient to cause significant inhibition, with maximal inhibition observed after 4 h. Northern (RNA) blot analysis indicated that mRNA expression for IL-1 alpha and IL-1 beta in response to C. albicans stimulation was also down-regulated in GM-CSF-cultured monocytes exposed to LAK cells. Interestingly, freshly isolated, Percoll gradient-purified large granular lymphocytes did not suppress antigen presentation in GM-CSF-treated monocytes. Another important finding was the inability of LAK cells to suppress the ability of freshly isolated or gamma interferon-cultured monocytes, which are resistant to LAK cell-mediated lysis, to present antigen to T cells. In contrast, IL-3 was similar to GM-CSF in inducing LAK cell susceptibility in monocytes. Taken together, these results indicated that IL-2 can induce LAK cells to down-regulate antigen presentation function in a select set of monocytes that have been activated by colony-stimulating factor (GM-CSF and IL-3) but not by gamma interferon. LAK cells may therefore play an important role in regulation of monocytes and their function, depending on their differentiation state.

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

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-h 51Cr-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.

Ultrastructural characteristics of lymphokine-activated killer cells of the rat in comparison with natural killer cells.

Lymphokine-activated killer (LAK) cells with a broad spectrum of tumor cell killing have been reported to be related to natural killer (NK) cells morphologically and phenotypically. We here examine the ultrastructural characteristics of LAK cells of the rat, comparing them to those of normal and OK-432-activated NK cells. Results show that, five days after the culturing of spleen lymphocytes with human recombinant interleukin-2, there were induced LAK cells, which were large granular lymphocytes and had a cytotoxic capacity against NK-resistant P-815 tumor cells. They were larger in size than NK cells and richer in cell organelles such as ribosomes, rough endoplasmic reticulum, a Golgi apparatus, granules and vesicles. The granules of LAK cells were shown to be related to multivesicular bodies as those of NK cells; they included multivesicular bodies, fully dense granules and intermediate forms between them. The average numbers and sizes of the granules and the proportion of multivesicular bodies and intermediate forms among the total granules were greater in LAK cells than in NK cells. The density of the small vesicles packed in multivesicular bodies and intermediate forms was much higher in LAK cells. At the contacting surface of the LAK cells bound to the target cells, exocytosis of multivesicular bodies was shown to occur. We recognized here two populations of LAK cells with different types of vesicles, one containing rod-cored vesicles and the other a new type of vesicles termed "demilune-cored vesicles". The latter vesicles were the same in size as the rod-cored ones and contained a dense core located eccentrically. Between these two populations of LAK cells, there was no difference concerning the profile of the dense granules. The present study indicates that, although LAK and NK cells share several ultrastructural features, the former show markedly enriched cell organelles, which indicate an accelerated metabolism of the cell for continuous proliferation.

Induction of lymphokine-activated killer cells 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.

Regional transarterial infusion of autologous lymphokine-activated killer (LAK) cells for advanced renal cell carcinoma and its preliminary clinical result

We herein report the preliminary but appreciable results of regional transarterial infusion of 2 gravity subtypes of autologous lymphokine-activated killer (LAK) cells into the metastatic sites in combination with systemic recombinant interleukin-2 (rIL-2) administration in 3 patients with advanced renal cell carcinoma. Leukapheresis was performed once a week and peripheral blood lymphocytes were separated into 2 different subtypes by Percoll gradient centrifugation. These lymphocytes were incubated with rIL-2 for a few days to induce LAK cells. LAK cells were transferred to the metastatic lesions through cannula twice a week. A large iliac bone metastasis disappeared 3 months after the initial LAK cell therapy via a superior gluteal artery. A case of complete disappearance of psoas muscle and para-aortic lymphnode metastasis as well as partial regression of a lumbar bone metastasis was seen after lumbar arterial infusion treatment. Another case with brain metastasis showed a rapid exacerbation of brain edema after one week's LAK therapy. Our treatment modality seems to be worthwhile and promising for treatment of the advanced renal cell carcinoma.

In vitro induction of lymphokine-activated killer (LAK) activity in patients with neuroblastoma.

Therapy of disseminated neuroblastoma remains an unsolved problem in pediatric oncology. Therefore, new therapeutic approaches have to be developed for this malignancy. In this paper, we investigated the possibility of the in vitro generation and expansion of lymphokine-activated killer (LAK) cells in patients with disseminated neuroblastoma. Although the patients had very low Natural Killer (NK) activity, it was possible to induce LAK activity in peripheral mononuclear lymphocytes (PMNC) by incubation with Interleukin-2 (IL-2). Moreover, the PMNCs could be expanded up to 50-fold in the presence of Interleukin-2 while maintaining or even increasing their LAK activity. The target cells were neuroblastoma cell lines and, in one case, autologous neuroblastoma cells. Additionally, it was possible to induce LAK cell activity against autologous neuroblastoma cells in bone marrow-derived mononuclear cells.

IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte cultures.

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.