Improved therapeutic effects of interleukin 2 after the accumulation of lymphokine-activated killer cells in tumor tissue of mice previously treated with cyclophosphamide

Summary We investigated the combined effects of human recombinant interleukin 2 (IL-2) and cyclophosphamide (CY) on s.c. transplanted 3LL lung carcinoma in C57BL/6 mice. A total of 95% of the tumors were competely cured when CY (150 mg/kg, i.v.) was given on day 5 (5 days after tumor implantation) and IL-2 (5×10⁴ Jurkat Units/day, i.p.) was then combined with it between day 6 and day 15. CY alone brought about the complete regression of tumors, although 60% of the mice died of local recurrence and pulmonary metastasis; IL-2 alone had no therapeutic effect. Satisfactory effects from the combination of CY and IL-2 were also obtained by 5 days administration of IL-2 between days 11 and 15, initiated 6 days after CY treatment, but not by that given before CY (days 1–5) or 1 day after CY (days 6–10). No therapeutic effects from IL-2 were observed when it was combined with other types of chemotherapy that showed not therapeutic effects by themselves. Nor were we able to observe any transplantation resistance to the rechallenge of 3LL tumor in cured mice. We particularly examined the lymphokine-activated killer (LAK) cells as we suspected that these were responsible for the development of active effector cells in the treated mice. LAK cell activity in fresh spleen cells was detected in mice treated with IL-2 alone but not in untreated mice nor in those treated with CY alone or CY plus IL-2. The number of LAK precursor cells in the spleen had increased on day 8 and on day 13 in untreated mice with 3LL, as compared with the incidence in normal mice, while the number of cells had decreased by day 18. On the other hand LAK precursor cells were suppressed on day 8 and tended to recover thereafter in CY-treated mice. Adoptively transferred LAK cells were found to accumulate in CY-treated tumors 2.5 times more densely than in untreated tumors. The preferential accumulation of LAK cells that had been activated systemically by the appropriately timed administration of IL-2 in tumor tissue was followed by the improved effects obtained by combined treatment with CY and IL-2.Supported in part by Grants-in-Aid for Cancer Research from the Japanese Ministry of Education, Science and Culture and from the Japanese Ministry of Health and Welfare     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines

Summary The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon A/D (IFN), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6–18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN were administered three to five times weekly for 1–3 weeks, usually starting 2–5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN, IL-2 + IFN ± LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

Similarities and distinctions between murine natural killer cells and lymphokine-activated killer cells

Pretreatment of mice with rabbit anti-asialo GM1 removes both natural killer (NK) effector cells and NK cells responsive to interleukin 2 (IL-2). Spleen cells from these mice do possess normal lymphokine-activated killer (LAK) activity. Young mice (less than 3 weeks of age) do not have NK activity and do not possess IL-2-inducible NK effector cells. Similarly to anti-asialo GM1-treated mice, LAK cells can be generated from these mice. While these experiments indicate clear distinctions between a certain level of NK and LAK precursors, the distinctions are not as clear when analyzing mice congenitally deficient in NK cells. Beige mice which lack NK effector cells and IL-2-inducible NK cells also lack the ability to generate LAK cells. The relationships and differences between NK- and LAK-cell precursors and effectors are discussed.     

Importance in timing of cyclophosphamide on the enhancement of interleukin-2-induced cytolysis

Summary We investigated the in vivo effects of cyclophosphamide (CY) on interleukin-2(IL-2)-induced cytolytic function and spleen cell immunophenotype. Pretreatment of A/J mice with CY (25 mg/kg or 75 mg/kg) i.p. on days –10 and –15 followed by IL-2 (50 000 U i.p. on days 0 to +3) resulted in increased lysis of YAC-1 target cells compared to the group receiving IL-2 without previous CY therapy. In contrast, when CY was given on day -5, the cytotoxicity against YAC-1 was not enhanced. Phenotypic analysis of splenocytes obtained from mice treated with CY on day –10 or –15 revealed a relative decrease in L3T4- and Lyt2-positive T cells. In vivo depletion of natural killer (NK) cells by anti-asialoG_M1, prior to IL-2 therapy, abrogated the enhancing effect of CY on cytolysis while in vivo elimination of T cells by anti-L3T4 and anti-Lyt2 monoclonal antibodies did not, indicating that in the absence of T cell antigenic challenge, the increased cytolytic function after CY administration is probably mediated through NK cells. These findings provide evidence that CY may be used more effectively in IL-2-based immunotherapy protocols, if consideration is given to timing of CY and IL-2 administration.Supported in part by the Children's Cancer Research Fund, the Concern II Foundation, RO1-CA-21 737, NO1-AI-85 002 and a contract to the University of Minnesota from OncoTherapeutics Inc. Dr. E. Katsanis is supported by a fellowship from the Medical Research Council of Canada. Dr. P. M. Anderson is supported by a Clinical Oncology Career Development Award from the American Cancer Society. This is paper no. 550 of the Immunobiology Research Center     

Effect of a glucan,sizofiran, on natural-killer activity of 5-fluorouracil-treated murine bone marrow cells

Summary The number of bone marrow cells in C3H/He mice was reduced 3–4 days after treatment with 130 mg/kg intraperitoneal 5-fluorouracil (5-FU). Higher rates of spontaneous proliferation and natural killer (NK) activity, accompanied by an increase in asialoGM1-positive cells, were observed in treated mice. When sizofiran at a dose of 200 µg/animal was intramuscularly injected after 5-FU treatment, the rates of proliferation and NK activity of bone marrow cells were higher than with 5-FU alone. The cell number was not influenced by sizofiran alone. These results indicate that all precursors of the various mature cell types (including NK cells) differentiate and regenerate rapidly to replace cells damaged by 5-FU treatment, and that sizofiran has the potential to assist this recovery. These results suggest that administration of sizofiran after chemotherapy may be useful in cancer patients.     

cis-Diamminedichloroplatinum and 5-fluorouracil are potent inducers of the cytokines and natural killer cell activity in vivo and in vitro

It has been reported that certain chemotherapeutic agents exhibit effects that enhance the antitumor host responses in the patients with malignant diseases. In the present study, we investigated whether cis-diamminedichloroplatinum (cisplatin) and 5-fluorouracil (5-FU) may induce cytokines and effector cells with antitumor efficacy in vivo and in vitro. The cultivation of human peripheral blood mononuclear cells (PBMC) in the presence of cisplatin (0–1.0 μg/ml) or 5-FU (0–5.0 μg/ml) resulted in the significant augmentation of natural killer (NK) and lymphokine-activated killer (LAK) cell activities as well as generation of interferon (IFN) γ, tumor necrosis factor (TNF) α, β, interleukin(IL)-1β, IL-6 and IL-12 in vitro. In addition, all of these activities were almost completely neutralized by addition of anti-asialoGM1 antibody and complement (P < 0.05). In an in vivo model, the administration of anti-asialoGM1 antibody significantly shortened the survival time extended by the treatment with cisplatin or 5-FU (P < 0.05), both on nude mice bearing salivary gland tumors and on syngeneic MethA-tumor-bearing BALB/c mice. Furthermore, high levels of NK and LAK activities and significant increases of the numbers of cells positive for asialoGM1, IFNγ, TNFα, or IL-1β were detected in the spleen cells derived from animals given cisplatin or 5-FU as compared with those given saline (P < 0.001–0.05). These findings clearly indicate that cisplatin and 5-FU are potent inducers of several types of cytokines and effector cells carrying antitumor activity mediated by asialoGM1-positive cells (mainly NK cells) for the most part, and that these abilities are closely associated with the in vivo antitumor effect of these agents. Received: 23 July 1998 / Accepted: 10 September 1998     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Growth of MCF-7 human breast carcinoma in severe combined immunodeficient mice: Growth suppression by recombinant interleukin-2 treatment and role of lymphokine-activated killer cells

Summary The severe combined immunodeficient (SCID) mouse, lacking functional T and B lymphocytes, has been considered by many groups to be a prime candidate for the reconstitution of a human immune system in a laboratory animal. In addition, this immuno-deficient animal would appear to have excellent potential as a host for transplanted human cancers, thus providing an exceptional opportunity for the study of interactions between the human immune system and human cancer in a laboratory animal. However, because this animal model is very recent, few studies have been reported documenting the capability of these mice to accept human cancers, and whether or not the residual immune cells in these mice (e.g. natural killer, NK, cells; macrophages) possess antitumor activities toward human cancers. Thus, the purpose of this study was (a) to determine whether or not a human breast carcinoma cell line (MCF-7) can be successfully transplanted to SCID mice, (b) to determine whether or not chronic treatment of SCID mice with a potent lymphokine (recombinant interleukin-2, rIL-2) could alter MCF-7 carcinoma growth, and (c) to assess whether or not rIL-2-activated NK cells (LAK cells) are important modulators of growth of MCF-7 cells in SCID mice. To fulfill these objectives, female SCID mice were implanted s.c. with MCF-7 cells (5 × 10⁶ cells/mouse) at 6 weeks of age. Six weeks later, some of the mice were injected i.p. twice weekly with rIL-2 (1 × 10⁴ U mouse^–1 injection^–1). Results clearly show that MCF-7 cells can grow progressively in SCID mice; 100% of the SCID mice implanted with MCF-7 cells developed palpable measurable tumors within 5–6 weeks after tumor cell inoculation. In addition, MCF-7 tumor growth was significantly (P <0.01) suppressed by rIL-2 treatment. rIL-2 treatment was non-toxic and no effect of treatment on body weight gains was observed. For non-tumor-bearing SCID mice, splenocytes treated in vitro with rIL-2 (lymphokine-activated killer, LAK, cells) or splenocytes derived from rIL-2-treated SCID mice (LAK cells) had significant (P <0.01) cytolytic activity toward MCF-7 carcinoma cells in vitro. In contrast, splenocytes (LAK cells) derived from tumor(MCF-7)-bearing rIL-2-treated SCID mice lacked cytolytic activities toward MCF-7 cells in vitro. No significant concentration of LAK cells in MCF-7 human breast carcinomas was observed nor did rIL-2 treatment significantly alter growth of MCF-7 cells in vitro. Thus, while rIL-2 treatment significantly suppressed growth of MCF-7 breast carcinomas in SCID mice, the mechanism of this growth suppression, albeit clearly not involving T and B lymphocytes, does not appear to be mediated via a direct cytolytic activity of LAK cells toward the carcinoma cells. However, rIL-2-activated SCID mouse splenocytes (LAK cells) do possess the capability of significant cytolytic activity toward MCF-7 human breast carcinoma cells. Thus, treatment of SCID mice with a potent lymphokine (rIL-2) induces a significant antitumor host response, a response that does not involve T and B lymphocytes and appears not to involve NK/LAK cells. This host response must be considered in future studies designed to investigate the interactions of reconstituted human immune systems and human cancers within this highly promising immuno deficient experimental animal model.     

Synergistic antitumor activity of interleukin-2 and cimetidine against syngeneic murine tumor

Summary Cimetidine, an H2 histamine receptor antagonist, is a potent immunomodulating agent, which acts by inhibiting suppressor T lymphocyte function. The present work investigated the effect, if any, of cimetidine on interleukin-2 (IL-2)-induced natural killer (NK) and lymphokine-activated killer (LAK) cell activities, and on in vivo antitumor activity using syngeneic colon 26 adenocarcinoma as the model. Mimicking the clinical conditions, all in vitro experiments were evaluated with the splenocytes prepared from tumor-bearing BALB/c mice. Ten days after subcutaneous inoculation of tumor cells (5 × 10⁵), animals were treated intraperitoneally daily with phosphate-buffered saline (PBS), cimetidine (2 mg kg^–1 day^–1), IL-2 (300 000 IU/day), or cimetidine plus IL-2 for 7 consecutive days. The treatment of IL-2 plus cimetidine increased NK and LAK cell activities significantly and synergistically at the end of the treatment (i.e. on day 18) as well as 1 week after the treatment (i.e. on day 25), in comparison with those of the control groups (PBS, cimetidine alone, IL-2 alone). Also, in vivo antitumor activity, as analyzed by a Kaplan-Meier life table with the log-rank test, revealed a significantly prolonged survival in the group treated with IL-2 plus cimetidine compared to the control groups. Phenotyping performed on the murine splenocytes on day 18 indicated a significant reduction in Lyt2-positive cells in the cimetidine-treated group in comparison with the PBS group. A significant increase in asialo GM1-positive cells and IL-2-receptor-positive cells was detected in the group treated with IL-2 plus cimetidine in comparison with the PBS and IL-2 control groups. Therefore, this study indicates a synergistic enhancement of IL-2-induced NK and LAK cell activities in tumor-bearing hosts by cimetidine, a noncytotoxic inhibitor of suppressor T function, and a significantly prolonged survival of tumor-bearing animals treated by IL-2 plus cimetidine. It also suggests the clinical potential of combination therapy of IL-2 with cimetidine.     

Differential sensitivity of cytotoxic T lymphocytes and lymphokine-activated killer cells to inhibition by L-ornithine

The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/2 mixed leukocyte cultures (MLC) by the amino acid L-ornithine (Orn) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from 10-1000 U/ml and measuring cytolytic activity against two tumor targets (P815 and YAC-1) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CD8- and ASGM1+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2.     

Lymphokine-activated killer cells are rejected in vivo by activated natural killer cells

A 4-h in vivo cytotoxicity assay was used to study the fate of implanted IL-2-generated, lymphokine-activated killer (LAK) cells in mice undergoing an activated NK cell response. 125Iododeoxyuridine-labeled LAK cells were rejected from selected organs of C57BL/6 mice infected with lymphocytic choriomeningitis virus or treated with IL-2 or the IFN inducer poly I:C. This rejection was abrogated by the selective depletion of NK cells with antibodies to asialo-GM1 and NK1.1 Ag. Similar results were noted when LAK cells were generated from the spleens of B and T cell-deficient severe combined immunodeficiency mice and when LAK cells were implanted into severe combined immunodeficiency mice. These data indicate that NK cells activated by virus infections or by IL-2 infusions directly or indirectly eliminate implanted LAK cells. Because LAK cells are used in the treatment of certain human cancers, the strategy of accompanying this therapy with IL-2 infusions should be reassessed in light of these results.     

Influence of the donors' clinical status on in vitro and in vivo tumor-cytotoxic activation of interleukin-2-exposed lymphocytes and their circulation in different organs

Summary In-vitro-generated lymphokine-activated killer (LAK) cells of BALB/c mice, bearing the syngeneic colon carcinoma C-26 for 7 days, were as efficient as those from normal mice in lysing C-26 cells whereas LAK cells from 14-day tumor-bearing and 5- and 14-day tumor-resected animals had a lower C-26 cytotoxicity. The level of C-26 lysis returned to normal values 30 days after surgery. To identify the best source of LAK cells in vivo, groups of normal mice were treated with 10⁴, 3×10⁴ or 10⁵ U/day of interleukin 2 (IL-2) for 7 days intraperitoneally (i. p.) or intravenously (i. v.) (3×10⁴ dose only). The highest lysis on C-26 was obtained from peritoneal exudate cells of mice given 3×10⁴ and 10⁵ U whereas spleen cells were lytic only when taken from mice treated with 10⁵ U IL-2. Peripheral blood lymphocytes lacked any cytotoxicity except for the group of mice which received IL-2 i. v. The kinetics of in vivo LAK activation in different organs showed a peak of anti-(C-26) lytic activity at day 5 in peritoneal exudate cells and spleen cells of mice given IL-2 for 5 days whereas administration of LAK cells alone had no effect; IL-2 plus LAK cells gave a lower peak of LAK activity as compared with IL-2 alone. A lower level of in vivo LAK activation was found in mice whose tumor was resected 5 days before; such impairment was evident even 14 days after surgery. Homing experiments were carried out with i. v. injected ⁵¹Cr-labelled LAK cells in normal or tumor-resected mice. In normal mice the highest radioactivity at 30 min was found in the lungs; liver and spleen also showed high radioactivity whereas blood had a negligible amount of radioactivity. Radioactivity declined rapidly in lungs (less than 10% after 24 h) while remaining at appreciable levels in the liver after 24 h and 48 h; spleen showed constant levels of 12%–15%. Homing of LAK cells was altered in mice receiving IL-2 i. p. for 5 days with slower and lower radioactivity peaks in the lung and higher levels in liver. In tumor-excised mice lower levels of radioactivity were found in lungs. These results show that: (a) alterations in LAK activity occur in early-tumor-resected and large-tumor-bearing animals; (b) the route of IL-2 administration is critical in LAK activation in vivo; (c) treatment with IL-2 modifies LAK homing.This study was in part supported by grant no. 87.01565.44 of the Finalized Project Oncology of CNR (Rome, Italy)     

Ultrastructural and functional studies of the interaction between IL-12 and IL-2 for the generation of lymphokine-activated killer cells

IL-12 promotes generation of LAK activity in short-term-cultured NK cells, but information on the structure and function of IL-12-induced LAK cells is not yet available. The latter issues have been here investigated with emphasis on interactions between IL-12 and IL-2. Peripheral blood mononuclear cells (MNC) exposed to IL-12 for 5-7 days displayed a decrease in the amount and density of the matrix of large granular lymphocyte (LGL)-associated granules. In cells cultured with IL-12 and IL-2 for 5-7 days, empty vacuoles were predominant and the electron-dense matrix was scanty. In MNC incubated with IL-2 for 5-7 days, most granules were loaded with electron-dense matrix. IL-12 and IL-2 displayed an additive effect on LAK cell cytotoxicity until approximately 48 h in culture which was followed by a sharp decline. Immunocytochemical and biochemical studies demonstrated that MNC cultured for 5-7 days with IL-12 and IL-2 displayed downregulated perforin expression and upregulated granzyme B expression. Fas ligand expression was virtually undetectable in MNC cultured for 5-7 days with or without cytokines. It appears that perforin downregulation plays a major role in the reduced cytotoxicity of MNC cultured with IL-12 and IL-2 for 5-7 days.     

In vitro generation of natural killer cells from SJL/J bone marrow precursors

The development of natural killer (NK) cells from undifferentiated bone marrow (BM) precursors of low-NK-reactive SJL/J mice was studied. Results indicate that BM cells of untreated mice are not able to generate NK effector cells in cultures supplemented with recombinant interleukin-2 (IL-2). On the other hand in the presence of IL-2, NK cells are generated in cultures of BM from mice pretreated with 5-fluorouracil (5-FU, 150 mg/kg iv 4 days before harvesting), a treatment which has been shown to eliminate more differentiated but spare less differentiated BM precursors. The 5-FU resistant BM progenitor is asialoGM1-, Thy.1+, Lyt.1- and Lyt.2-. The cells generated by culturing with IL-2 are asialoGM1+, Thy.1+, Lyt.5+, Lyt.1-, Lyt.2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2 receptor (IL-2/r) antibodies. These studies demonstrate that it is possible to generate NK effectors from SJL/J BM cells by in vitro culturing with IL-2.     

Murine trophoblast can be killed by lymphokine-activated killer cells

The ability of fetal trophoblast cells in the placenta to resist cell-mediated lysis may be important for successful pregnancy. Previous studies in this laboratory demonstrated that cultured midterm mouse trophoblast cells are not susceptible to allospecific CTL generated by standard in vitro protocols, to antibody-dependent cell-mediated cytotoxicity, or to naive or IFN-activated NK cells, despite expressing the requisite target structures. However, we now report that murine trophoblast can be killed, in a non-MHC-specific manner, by LAK cells. Normal mouse spleen cells cultured for 4 days in IL-2-containing lymphokine preparations characteristically killed both NK-sensitive (YAC-1) and NK-resistant (EL4, P815) target cells, and mediated significant lysis of both cultured and freshly isolated trophoblast cells (35 to 55%, E/T 100/1). Pretreatment of the LAK cells with anti-ASGM1 antibody and C markedly reduced the lysis of trophoblast and YAC-1 targets, suggesting that the responsible cells belonged to the NK lineage. The ability of IL-2-activated NK cells to kill midterm murine trophoblast cells was confirmed using a population of highly lytic NK cells generated by culturing spleen cells from severe combined immunodeficiency mice in 500 U/ml rIL-2 for 5 days. These effector cells killed YAC-1, EL4 and P815 target cells at much lower E/T ratios than was achieved with the normal splenic LAK cells, and mediated significant lysis of both freshly isolated (45 to 50%, E/T 20/1) and cultured trophoblast cells (68 to 76%, E/T 20/1). The susceptibility of trophoblast to LAK cells and IL-2-activated NK cells supports the need for suppressor mechanisms regulating IL-2 activity at the maternal-fetal interface.     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

Functional studies on the precursors of human lymphokine-activated killer cells

Human peripheral blood lymphocytes cultured for 4 days in the interleukin 2 (IL-2)-containing cell-free supernatant of the MLA144 cell line (MLA144CM) are cytolytic to NK-susceptible and NK-resistant tumor target cells. This lymphokine-activated killer (LAK) activity is dependent on IL-2 as development of LAK activity is inhibited in the presence of a monoclonal antibody (MoAb) reacting with the IL-2 receptor (anti-Tac). Addition of cyclosporin A (CyA) to mixed lymphocyte cultures inhibits the development of allospecific cytotoxic activity and inhibits the development of IL-2 responsiveness. However, development of LAK activity is unaffected by the inclusion of CyA in the cultures, showing that the LAK precursor can be functionally distinguished from the allospecific cytotoxic precursor cell. Development of LAK activity does not require mature NK cells as shown by the generation of LAK activity from NK inactive human thymocytes and lymph node cells. In addition, depletion of NK activity from human PBL does not impair the development of LAK activity.