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.     

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.     

Recombinant interleukin 2 stimulates in vivo proliferation of adoptively transferred lymphokine-activated killer (LAK) cells

We previously reported that the adoptive transfer of lymphokine-activated killer (LAK) cells plus repetitive injections of recombinant interleukin 2 (IL 2) produced a marked reduction in established pulmonary metastases from a variety of murine sarcomas. The requirement for the exogenous administration of IL 2 prompted a subsequent examination of the role of IL 2 in the in vivo function of transferred LAK cells. The in vivo proliferation and migration patterns of lymphoid cells in C57BL/6 mice were examined after i.v. transfer of LAK cells alone, i.p. injection of IL 2 alone, or the combination of LAK cells and IL 2. A model for in vivo labeling of the DNA of dividing cells was used in which mice were injected with 5-[125I]-iodo-2'-deoxyuridine (125IUdR) and, 20 hr later, their tissues were removed and were counted in a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. In animals given LAK cells alone, the lungs and liver demonstrated little if any uptake of 125IUdR above saline-treated controls (PI = 2.5 and 0.8, respectively, on day 5), whereas the same organs of mice receiving 6000 U of IL 2 alone displayed higher radiolabel incorporation (PI = 7.1 and 5.9, respectively). When mice were given LAK cells plus 6000 U of IL 2, their tissues showed an additional increase in 125IUdR uptake. In the spleen, kidneys, and mesenteric lymph nodes, IL 2 treatment alone (6000 U) produced elevated PI values that were not, however, additionally increased if LAK cells were also administered. To separate the stimulatory effects of IL 2 on host lymphocyte proliferation from similar IL 2 effects on injected LAK cells, these studies were repeated in mice immunosuppressed by 500 rad total body irradiation. Pre-irradiation of the host sufficiently reduced endogenous lymphoid expansion stimulated by IL 2 so as to allow the demonstration that IL 2 also induced the proliferation of the transferred LAK cells. A variety of studies confirmed that the injected LAK cells were actively proliferating in tissues in vivo under the influence of IL 2. Substitution of "normal" LAK cells with fresh and cultured (without IL 2) splenocytes, or irradiated LAK cells did not result in increased 125IUdR uptake in tissues. Histologic studies corroborated the findings of the 125IUdR incorporation assays and revealed extensive lymphoid proliferation in irradiated mice receiving LAK cells plus IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Antitumour efficacy of lymphokine-activated killer cells loaded with ricin against experimentally induced lung metastases

Summary Adoptive transfer of tumour-specific T lymphocytes loaded with ricin into tumour-bearing mice exerts a transient therapeutic effect against locally induced tumours [Cerundolo et al. (1987) Br J Cancer 55: 413]. As transferred cells preferentially locate in the lung, we studied the therapeutic effect of ricin-loaded, lymphokine-activated killer (LAK) cells on lung metastases induced by M4 or B16-F1 (F1) tumour cell injection. In vitro studies demonstrated that ricin-treated LAK cells were about 100-fold more efficient than untreated LAK cells in inhibiting growth of the ricin-sensitive M4 tumour cell line. This effect was most likely due to the released ricin, as treated and untreated LAK cells inhibited the relatively toxin-resistant F1 cell line to the same extent. Ricin treatment did not alter the tissue distribution of intravenously (i.v.) injected LAK cells, which selectively localized in the lung early after inoculation, whether or not metastases were present. Adoptive transfer experiments showed that ricintreated LAK cells were significantly more efficient than untreated LAK cells in inhibiting M4- but not F1-induced lung metastases. These results indicate that LAK cells are able to deliver a therapeutic concentration of antineoplastic compounds directly to the lung.Research activities were partially developed in relation to a contract with the National Program of Pharmacological Research (Rif. 078606), by the Italian Consortium for Antitumoral Vectors (C.I.V.A) for the Italia Ministry of University and Scientific and Technological Research     

Effect of interleukin 2 on fetal thymocytes in organ cultures: generation of lymphokine-activated killer cells

Cells with cytolytic activity can be detected in mouse fetal thymic lobes cultured in the presence of interleukin 2 for 6 days. The lymphokine-activated killer cells from 14-day fetal thymic lobes are relatively resistant to treatment with anti-Ly-2 antibody and complement (CD8-) but sensitive to anti-Thy-1 and complement treatment (Thy-1+). They display major histocompatibility complex-unrestricted killing, lysing both syngeneic and allogeneic tumor cells, but will not lyse human xenogenic target cells. Low levels of cytotoxic activity can be detected in thymic lobes from Day 12-13 embryos and this activity increases with embryonic age. While the events which lead to the inhibition of normal maturation of fetal thymocytes by inclusion of IL-2 in fetal thymus organ cultures are unknown, the appearance of cytotoxic cells raises the question of whether they are involved in the normal intrathymic cell death process.     

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     

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.     

A regimen of surgical adjuvant immunotherapy for cancer with interleukin 2 and lymphokine-activated killer cells

We designed a unique regimen of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin 2 (rIL-2) for application with surgical adjuvant therapy of cancer. The regimen features the prolonged (6 consecutive days) s.c. administration of low-dose rIL-2 and the transfer ofex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of surgical operation. According to this regimen, 5 patients with primary lung cancer received immunotherapy about 2 weeks after surgery (pulmonary lobectomy). Clinical toxicities included fever(5/5), fatigue(5/5), slight(< 5%) weight gain(5/5), increase of pleural effusion at the lobectomy site(2/5), and edema formation(1/5). All toxicities reversed within 4 days after the completion of therapy. Rebound lymphocytosis after therapy ranged from 2.4 to 5.5-fold (mean, 4.3-fold) over the baseline. Peripheral blood lymphocytes obtained during this lymphocytosis exhibitedin vitro LAK activity in 4 of 5 patients. Thus, the regimen is considered to be well-tolerable and immunologically active in regard to the postoperative state of the patients.     

Adherent lymphokine-activated natural killer cells in normal and severe combined immunodeficiency mice: large granular lymphocytes with natural killer cell phenotype and high cytolytic activity

Plastic-adherent lymphokine-activated natural killer (LANK) cells were generated from nylon wool-nonadherent murine splenocytes cultured in recombinant interleukin-2 (IL-2). Under such conditions, adherent lymphokine-activated killer cells capable of killing natural killer (NK)-resistant targets were not generated. Adherent LANK cells proliferated rapidly and closely resembled NK cells in their morphology, cytotoxic reactivity, and surface marker expression. Mice with severe combined immunodeficiency (scid) were used to generate adherent LANK cells to define the role of T cells in LANK cell development. Scid lymphocytes responded to IL-2 by becoming adherent LANK cells with potent NK-like activity, suggesting that soluble lymphokines other than IL-2 that may have been produced by T cells were not required for the generation of LANK cell activity in mice.     

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.     

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     

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

Modulation of human natural killer cell activity by recombinant human interleukin 2

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range of 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes, and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.     

The anti-tumour efficacy of human recombinant interleukin 2

Summary Experiments were designed to test what percentage of experimental MC-induced murine sarcomas were sensitive to the local tumour inhibitory effect of IL-2 and whether any correlation existed between the sensitivity of these sarcomas to the immunotherapeutic effect of IL-2 and their susceptibility to the cytolytic effect of IL-2-activated killer cells. It was found that the sensitivity of MC-induced sarcomas to local IL-2 immunotherapy was a general phenomenon. Repeated peri-tumoural injections of RIL-2 inhibited the growth of five (MC11, MC13, MC14, MC15, MC16) out of six sarcomas in syngeneic mice. The sixth murine sarcoma (MC12) was found to be resistant to the tumour inhibitory effect of IL-2. Similarly, five (MC11, MC13, MC14, MC15, MC16) out of six murine sarcoma cell lines were sensitive to the cytolytic effect of IL-2-activated syngeneic killer spleen cells when examined in vitro, whereas the sixth (MC12) sarcoma cell line was resistant. These results suggest that LAK cells represent the effector cell mechanism responsible for the anti-tumour efficacy of local IL-2 immunotherapy and that in vitro testing of sensitivity to the LAK cell-mediated cytolysis may be used to detect tumours responding to IL-2 immunotherapy in vivo.Abbreviations IL-2 interleukin 2 - RIL-2 human recombinant interleukin 2 - LAK lymphokine-activated killer - MC 3-methylcholanthrene - B10 C57BL/10ScSnPh - MSV murine sarcoma virus (Harvey) - MEM minimal essential medium     

Influence of T cells on the expression of lymphokine-activated killer cell activity and in vivo tissue distribution

Lymphokine-activated killer (LAK) cells are currently being evaluated in several cancer centers for the immunotherapy of patients with a variety of cancers. Understanding the in vivo distribution of LAK cells should help to optimize their antitumor efficacy. As a model system to examine this issue, nylon wool column-passed rat lymphocytes were cultured in the presence of rIL-2 for 1 and 2 days. The resulting cells were divided into two populations; one that adhered to the plastic flasks and the second which did not adhere. The adherent cells were found to be highly cytotoxic against NK-sensitive and NK-resistant targets, whereas the nonadherent cells were unable to kill NK-resistant targets unless T cells were removed from this population. These results indicate that T cells present in IL-2 activated bulk splenocytes may interfere with the activity of LAK cells. Adherent or nonadherent LAK cells were evaluated for their pattern of in vivo distribution after i.v. inoculation. These cells were found to display a restricted pattern of distribution, localizing mainly in the lungs at 2 h after i.v. injection but redistributing into the liver and the spleen by 24 h. LAK cells were rarely recovered from the lymphoid tissues, including the peripheral lymph nodes and the mesenteric lymph nodes. However, if T cells were not removed from the LAK cell population, some radioactivity was recovered from the peripheral and mesenteric lymph nodes. Fractionation of 2 day-activated, nonadherent population on discontinuous Percoll resulted in the enrichment of large granular lymphocyte (LGL)/LAK activity in low density fractions (42% and 45% Percoll), whereas high density fraction (70% Percoll) contained T cells which showed no cytolytic activity. Upon transfer into syngeneic rats, the 42% fraction showed typical LAK migration. In contrast, the 70% fraction showed typical T cell migration. What is more important, removal of the granulated cells resulted in a population which have no granules and resemble large agranular lymphocytes known to be pre-LGL/LAK cells. Large agranular lymphocytes showed a pattern of distribution different from both T and LGL/LAK cells.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.     

Susceptibility of Adenovirus 2-transformed rat cell lines to natural killer (NK) cells: direct correlation between NK resistance and in vivo tumorigenesis

The susceptibility to natural killer (NK)-mediated cell lysis of Adenovirus type 2 (Ad2)-transformed rat embryo fibroblast cell lines, which differed markedly in tumorigenic potential in vivo (T2C4 greater than F19 greater than F17), was investigated by using NK effector cells from F344 rat or athymic nude rat spleens. A comparison of the degree of NK-mediated lysis obtained with these tumor cell targets suggested a direct relationship between the resistance of a cell to NK cell lysis and its potential to form tumors in vivo. The cells were lysed in the following order of increasing susceptibility: T2C4 less than F4 less than F19 less than F17. Whether T cells or macrophages played a significant role in the observed lytic activity was determined by treating the NK effector cell population with anti-rat T cell serum (alpha T) and complement or by depletion of macrophages after binding to a glass bead column and treatment with carbonyl iron. A series of clonal sublines derived from the parental F17 and F4 cell lines further strengthened this relationship between tumorigenesis and resistance to NK-mediated cell lysis. Tumorigenic subclones from the non-tumorigenic F17 parental cells were demonstrated to be comparatively resistant to NK-mediated lysis. Tumorigenic subclones from tumorigenic F4 parental cell population showed a susceptibility to NK-mediated cell lysis virtually identical to the parental F4 cells. The implication of these results are discussed.     

In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.     

Immunotherapy of intraperitoneal cancer with interleukin 2 and lymphokine-activated killer cells reduces tumor load and prolongs survival in murine models

The control of malignancy disseminated within the peritoneal cavity is an important problem in the management of low-grade gastrointestinal and ovarian neoplasms. A model of peritoneal carcinomatosis in the mouse was used to investigate the potential of lymphokine-activated killer (LAK) cells and exogenous interleukin 2 (IL-2) to control intraperitoneal tumor. LAK cells are splenocytes activated in vitro by IL-2. C57BL/6 mice were injected intraperitoneally with a lethal inoculum of syngeneic MCA-105 tumor. Three days later, the established tumor was treated with adoptively transferred LAK cells and/or exogenous IL-2 administration. LAK cells alone were ineffective in reducing intraperitoneal tumor. Administration of IL-2 alone resulted in limited tumor reduction. Treatment with exogenous IL-2 in conjunction with LAK cells resulted in the greatest reduction of intraperitoneal tumor. The larger the number of LAK cells given, the greater the reduction in tumor. Frequent intraperitoneal bolus administration of IL-2 was more effective than a single daily intraperitoneal injection and intraperitoneal administration of IL-2 and LAK was more effective than systemic treatments. Marked prolongation of life was seen in mice treated with LAK cells plus exogenous IL-2. We conclude that intraperitoneal LAK cells plus exogenous IL-2 is an effective treatment regimen for reducing intraperitoneal tumor in this murine model.