Effect of anticancer drugs on the release of interferon γ in vitro

Summary Some conventional and experimental anticancer drugs were tested for their effect on concanavalin-A-induced interferon release from rat splenocytes in vitro. When 2.5 × 10⁶ rat splenocytes/ml, stimulated with 1 µg/ml concanavalin A, were incubated with various non-cytotoxic doses of the vinca alkaloid vincristine, there was an inhibition of the release of interferon in culture supernatants. The antitumour antibiotics bleomycin and Adriamycin, alkylating agents 4-hydroperoxycyclophosphamide and mafosfamide, and the immunoactive peptides FK 156 and FK565 did not affect the release of interferon under similar conditions. However, cyclosporin A, in similar experiments, markedly inhibited the release of interferon .     

Intrapleural instillation of interferon γ in patients with malignant pleurisy due to lung cancer

 The effect of intrapleural instillation of recombinant human interferon γ (IFNγ) at increasing doses of (1–12) × 10⁶ U was examined in six patients with cytologically positive pleural effusion due to lung cancer. Intrapleural instillation was repeated up to three times. Clinically, no reaccumulation of pleural effusion was observed in one patient and disappearance of lung cancer cells from the pleural effusion was seen in two other patients. No severe side-effects were observed. Considerable levels of IFNγ remained in the pleural effusion as well as in patients’ serum up to 7 days after instillation of 2 × 10⁶ U and higher doses. The total cell number showed a transient decrease on day 1 of therapy. Levels of pro-inflammatory cytokines, such as tumor necrosis factor α, interleukin(IL)-1β and IL-6, in the pleural effusion remained almost stable after IFNγ instillation. On the other hand, intrapleural IL-1 receptor antagonist levels were remarkably elevated by the instillation of IFNγ. IL-2- and IL-12-inducible killer activity of pleural mononuclear cells tended to increase slightly. Despite the inability of IFNγ to control pleural effusion in this treatment schedule, IFNγ instilled by an intrapleural route had a potential local antitumor activity. Moreover, since IFNγ persists in pleural effusions for a long time after a single instillation, such a therapy in combination with other fibrogenic biological response modifiers can be promising. Received: 28 February 1997 / Accepted: 23 July 1997     

A phase I trial with recombinant interferon γ (Roussel UCLAF) in advanced cancer patients

Summary A total of 29 patients with advanced malignancy were treated with recombinant interferon (rIFN, specific activity = 2.10⁷ units/mg, purity >95%) given by intravenous bolus at doses escalating from 0.01 mg/m² to 5 mg/m² (2 × 10⁵–10⁸ IU/m²) in nine successive steps (at least 3 patients/step). Injections of rIFN were repeated every 72 h for 15 days. Toxicity was evaluated according to the WHO scale. Fever and chills occurred in all patients treated without clear dose effect. Nausea and vomiting appeared at the fifth dose level and their frequency seemed to be dose-related. Cardiovascular side-effects (first-degree atrioventricular reversible block) were observed at the 2 mg/m² and 5 mg/m² levels (3 patients). Hematological toxicities were mild (2 grade 1 and 1 grade II cases of granulocytopenia). Minor biological modifications included a transitory rise in hepatic enzymes (12 patients), which correlated with the presence of liver metastasis. Hypocholesterolemia was observed in 18 patients. The appearance of antibodies against rIFN was not detected. One partial clinical response was observed in a patient receiving 2 mg/m². During rIFN therapy this patient had the highest scores in this series for peripheral T lymphocytes with an activated phenotype (HLA DR⁺, TAC⁺) = 15% and for natural killer (NK) cells (NKH1, Leu19⁺) = 17%. rIFN appears as a well-tolerated and promising therapeutic agent with toxicities and mode of action probably distinct from IFN and .     

Cytogenetic characteristics of patients with signs and symptoms of myelodysplastic syndromes in the State of Pará, Brazil

The myelodysplastic syndromes (MDS) are clonal hematopoietic diseases characterized by medullary dysplasia, cytopenias, and frequent evolution to acute myeloid leukemia. In 1982, the French-American-British (FAB) group proposed a classification for the MDS, based on morphological characteristics of peripheral blood and of the bone marrow. Later, cytogenetics proved to be a useful tool for the refinement of prognosis, through the use of the International Prognosis Score System (IPSS), as well as through evidence of clonality. Recently, the World Health Organization (WHO) proposed a new classification for the MDS, based on significant modifications of the FAB proposal, with the inclusion of chromosome analysis. A cytogenetic analysis was made of 17 patients with symptoms of MDS in the State of Para, based on WHO recommendations, and application of the IPSS. Good metaphases were obtained for 13 patients; 12 had a normal karyotype and only one had a clonal abnormality, del(3)(p25). The genes related to neoplastic processes that have been mapped to 3p are: XPC in 3p25.1 and FANCD2 and VHL in 3p25-26. Four patients had classic symptoms of MDS; in the rest the possibility of MDS was excluded or several months of observation before diagnosis were recommended. Among those with MDS, it was not possible to apply IPSS and WHO recommendations, because fundamental data were lacking, specifically the medullary blast and ring sideroblast counts. We advocate the implementation of routine cytogenetic analyses for the study of MDS, especially in patients with moderate hematopoietic dysplasia.     

Antitumor effect of combination of murine recombinant interferon β, murine recombinant interferon γ and human recombinant interleukin-2 in MethA-bearing mice

Summary We have previously reported that the combination of murine recombinant interferon (Mu-rIFN) with murine recombinant interferon (Mu-rIFN) provided greater inhibition of tumor growth than did each one alone in MethA-bearing mice. In the present study the effect of addition of human recombinant interleukin-2 (Hu-rIL-2) to the combination of Mu-rIFN with Mu-rIFN on tumor growth in BALB/c mice bearing syngeneic MethA fibrosarcoma was examined. Low doses of Hu-rIL-2 (5 × 10³ U or 5 × 10⁴ U at 3-day intervals) showed no antitumor activity, while a high dose of Hu-rIL-2 (5 × 10⁵ U) showed profound growth inhibition. The administration of IL-2 (ranging between 5 × 10³ U and 5 × 10⁵ U) in addition to the combination of IFN and IFN showed more augmented antitumor effects in a dose-dependent manner. Furthermore, the simultaneous administration of IL-2, IFN and IFN had more effective therapeutic activity, compared with the sequential administration of interferons and IL-2. These findings indicated that IL-2 in combination with IFN and was effective for cancer treatment.     

Neutralizing interferon β antibodies in melanoma patients treated with recombinant and natural interferon β

The incidence and clinical significance of therapy-induced neutralizing interferon (IFNß) antibodies was studied in a group of 21 melanoma patients treated with natural IFNß and 7 patients treated with recombinant IFNß. They were treated subcutaneously with 3×10⁶ IU three times per week in an adjuvant open trial for 24 weeks after surgical removal of all detectable metastases. Of the 21 patients treated with natural IFNß, 95% developed significant levels of neutralizing antibodies after 24 weeks. In comparison, 28% of the 7 patients treated with recombinant IFNß developed neutralizing IFNß antibodies. Cross-reactivity of the antibodies could be demonstrated. Persistence of antibody titers was seen in 80% of the patients 24 weeks after cessation of treatment with natural IFNß. No correlation between the maximum antibody titers and the antibody persistence after cessation of therapy could be established. We detected a clear correlation between the formation of neutralizing antibodies and the decrease in 2-microglobulin and 2,5-oligoadenylate synthease and therefore the drop in biological activity. In this adjuvant trial there was no difference in relapse rate and time until relapse between antibody-positive and antibody-negative patients. No difference in clinical outcome could be established between the patients treated with natural IFNß and recombinant IFNß.     

Antitumor response to recombinant murine interferon γ correlates with enhanced immune function of organ-associated,but not recirculating cytolytic T lymphocytes and macrophages

The mechanism of therapeutic activity for recombinant murine interferon-(rMu IFN) in the treatment of metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the tumor-bearing organ) were tested after 1 week and 3 weeks of rMu IFN administration (i.v. three times per week). Natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocyte (CTL) activities against specific and nonspecific targets, and macrophage tumoristatic activity were measured. rMu IFN demonstrated immunomodulatory activity in most assays of immune function. The optimal therapeutic protocol of rMu IFN (2.5×10⁶ U/kg, three times per week) prolonged survival and decreased the number of pulmonary metastatic foci. This therapeutic activity was correlated with specific CTL activity from pulmonary parenchymal mononuclear cells (PPMC), but not from spleen or blood. Macrophage tumoristatic activity in PPMC also correlated with therapeutic activity, but activity in alveolar macrophages did not. However, therapeutic activity did not correlate with NK or LAK activity at any site. These results demonstrate that the optimal therapeutic protocol is the same as the optimal immunomodulatory dose for pulmonary CTL and macrophage activities. Furthermore, while immunological monitoring may help to optimize treatment protocols, current monitoring procedures that use readily accessible sites, particularly peripheral blood, may not accurately predict the therapeutic efficacy of biological response modifiers in clinical trials.By acceptance of this article, the publisher or recipient acknowledges the right of the US. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the articleThis research was sponsored by the DHHS, under contract N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government     

The in vitro development of cytotoxicity in response to granulocyte/macrophage-colony-stimulating factor or interferon γ in the peripheral blood monocytes of patients with solid tumors: Modulation by arachidonic acid metabolic inhibitors

Summary The capacity of granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interferon (IFN ) to elicit monocyte cytotoxicity in vitro in the peripheral blood monocytes of patients with solid tumors was investigated. The cytotoxicity elicited by IFN was significantly reduced in cancer patient monocytes compared to normal monocytes. The cytotoxicity elicited by GM-CSF, however, was comparable between cancer patient monocytes and normal monocytes, but was lower than that induced by IFN . Indomethacin, a cyclooxygenase inhibitor, significantly augmented IFN -elicited cytotoxicity in cancer patient monocytes, but not in normal monocytes. In contrast, indomethacin augmented GM-CSF-elicited cytotoxicity in both cancer patient monocytes and normal monocytes. Nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, was found to suppress cytotoxicity in response to IFN and GM-CSF in both cancer patient monocytes and normal monocytes. The addition of leukotrienes to NDGA-treated cultures restored the development of cytotoxicity. Thus there are differences in the in vitro response of cancer patient monocytes and normal monocytes to distinct biological activators. Furthermore, these responses can be manipulated by agents that modulate arachidonic acid metabolism.Supported in part by PHS grant CA 41741Fellow of the A. Onassis Foundation     

Synthesis,in vitro antimicrobial and cytotoxic activities of novel pyrimidine–benzimidazol combinations

A series of novel 4-substituted-2-{[(1H-benzo[d]imidazol-2-yl)methyl] thio}-6-methylpyrimidine derivatives were designed, synthesized and evaluated for their cytotoxic activities against four human cancer cell lines and inhibitory activities against five type culture strains in vitro. Some of synthetic pyrimidine–benzimidazol combinations showed good inhibitory activities against Stenotrophomonas maltophilia, especially compounds 7b and 7c. Compounds 7a and 7d exhibited enhanced activities against MGC-803 in vitro, when compared to 5-Fu.     

Phase I/II study of recombinant interferon α and γ in advanced progressive renal-cell carcinoma

Summary In vitro studies have documented the synergistic antiviral and antiproliferative activity of recombinant interferon (rIFN) and rIFN. Furthermore, rIFN is a strong immunomodulator with optimal effects at a relative low dose (0.1 mg/m²). On the basis of these observations, we began a phase I/II study with the combination of rIFN at 100 µg/m² (2 × 10⁶ IU/m²) and rIFN2c 6 µg/m² (2 × 10⁶ IU/m²), injected twice a week subcutaneously. In cases of stable or progressive disease we increased the dose of rIFN2c every 2 weeks by 6 µg/m² until the maximum tolerated dose was reached. A total of 32 patients with proven progressive renal-cell carcinoma were included. Of the 31 eligible patients, 21 were male and 10 female, their average age was 57.2 years (range 35–72), 28 had had nephrectomy, their median Karnofsky performance status was 90% (70%–100%), and their tumors were localized predominantly to visceral tissue. In 2, response was complete and in 6 it was partial, for a response rate of 25%. The disease had stabilized in 5 patients and progressed in 16. The median duration of partial response was 14 months (8–16 months); of 2 cases of complete response, 1 persists (23+ months), and the other suffered a relapse after 22 months. The median time to response was 24 weeks (18–24 weeks). The maximum tolerated dose of rIFN was 30 µg/m² (range of 6–36 µg/m²). Side-effects included those known to be associated with interferon treatment. One patient developed septicemia during a period with grade 4 leukopenia. Our study permits no conclusion regarding the additional value of rIFN.     

Synergism of synthetic acyltripeptide and its analogs with recombinant interferon γ for activation of antitumor properties of human blood monocytes

Summary Human blood monocytes freshly isolated by centrifugal elutriation from healthy volunteers were not cytotoxic to allogeneic A375 melanoma cells, but they were activated to the tumoricidal state by incubation in vitro with FK-565, (heptanoyl--D-Glu-(L)-meso-,-A₂pm(L)-D-AlaOH), which is a synthetic acyltripeptide closely resembling cell wall peptidoglycan peptides of Streptomyces in structure. Among 11 different derivatives of FK-565, 7 analogs were more potent activators of monocytes for tumor cell killing than FK-565. The maximal expression of tumoricidal nonocytes was dependent on the concentration of FK-565 or its analogs added and the ratio of monocytes to target tumor cells. In a parallel experiment, a combination of a subthreshold concentration of FK-565 or its analogs (FR-42148 and FR-42149) and recombinant interferon (rIFN-) induced significant monocyte-mediated tumorcell killing, indicating that the effects of rIFN- and acyltripeptide or its analogs in monocyte activation are synergistic. In contrast to rIFN-, recombinant rIFN-A and rIFN- had additive effects with acyltripeptide or its analogs in human monocyte activation. These results suggested that synthetic acyltripeptide and its analogs combined with rIFN- could be of clinical value for in situ activation of the tumoricidal activity of human blood monocytes responsible for eradication of cancer metastases.     

Phase I trial of adoptive immunotherapy of cancer patients using monocyte-derived macrophages activated with interferon γ and lipopolysaccharide

 Cells of the monocyte/macrophage lineage have shown antitumor activity in vitro and in murine models after activation with interferon (IFN) γ. In vitro data suggest an additional effect on macrophage antitumor activity when IFNγ is combined with endotoxin (lipopolysaccharides; LPS). In this study we treated nine cancer patients with a total of 62 MAK infusion cycles with autologous macrophages given intravenously (i.v.) after in vitro activation with IFNγ and LPS. Low-grade fever (WHO I/II) was the commonest side-effect. Chills, nausea, and headache were noted when the number of transfused macrophages exceeded 2×10⁸. One WHO IV toxicity occurred, consisting of hypotension after transfer of 3×10⁸ cells, defining this dose as the maximum cell number tolerated. After pretreatment with ibuprofen, however, the maximum cell number could be increased without reaching dose-limiting toxicity. The highest number of cells reinfused was 15×10⁸. Circulating interleukin(IL)-6 increased in a dose-dependent manner as did IL-1 receptor antagonist (IL-1RA) and IL-8. Tumor response consisted of one case of stable disease (12 weeks) in a patient with formerly progressing colorectal cancer and progressive diseases in eight patients. This study indicates that reinfusion of autologous LPS-activated macrophages upon pretreatment with ibuprofen is feasible and tolerated without major side-effects. Received: 22 May 1997 / Accepted: 2 October 1997     

Production of tumor necrosis factor α and interferon γ in interleukin-2-treated melanoma patients: Correlation with clinical toxicity

Summary Interleukin-2 (IL-2)-based immunotherapy regimens are accompanied by dose-limiting toxicity consisting of fever, tachycardia, chills and capillary leak syndrome. We hypothesized that the toxicity was caused by the induction and release of endogenous cytokines such as tumor necrosis factor (TNF) and interferon (IFN). We measured the serum levels of TNF and IFN in IL-2-treated melanoma patients and attempted a correlation with clinical toxicity. A total of 23 patients received either 6 × 10⁶ IU or 12 × 10⁶ IU Cetus IL-2/m² by i. v. bolus daily for 5 consecutive days on weeks 1, 3 and 5. Serum TNF and IFN levels were measured by enzyme-linked immunosorbent assay. Clinical toxicity was scored each day by objective measurements of hypotension, tachycardia, fever and chills/rigors. Clinical toxicity and IFN levels correlated nicely, peaking on the 5th day of each treatment cycle. The kinetics and magnitude of TNF production, however, were not predictable and did not correlate with either IFN or toxicity. Some patients had modest increases in TNF production while others had markedly increased levels during the second and third treatment weeks. Remarkably, these high levels persisted during nontreatment weeks and after completion of therapy. This clinical study demonstrates novel kinetics for immunoreactive TNF in IL-2 cancer patients, which do not correlate well with toxicity.This work was supported by NIH Grants CA 50 780 (J. E.) and CA 29 605, CA 12 582 (D. L. M.) and the U. C. Tobacco-Related Disease Research Program RT-62 (J. E.). J. E. is the recipient of an NCI Clinical Investigator Award (KO8-01360) and is a Dorothy and Leonard Straus Scholar at UCLA     

Interactions between interferon γ and retinoic acid with transforming growth factor β in the induction of immune recognition molecules

The cell-surface expression of major histocompatibility (MHC) antigens and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) is essential for target cell recognition by T lymphocytes. The expression of both classes of molecule is induced by various cytokines, notably interferon (IFN). Since transforming growth factor (TGF) has been recently reported to antagonise HLA-DR induction by IFN we have examined, using a number of murine and human cell lines, the effect of TGF on IFN-induced MHC class I and class II and ICAM-1 expression. All of the cell lines tested expressed elevated class I MHC following IFN treatment. Class II MHC induction was seen on most but not all of the cells, the exceptions being among a panel of human colorectal carcinoma cell lines. A striking difference between cells of different origin was noted in the response to TGF. TGF was found to antagonise IFN-induced class I and class II MHC expression on C3H 10T1/2 murine fibroblasts, early-passage BALB/c mouse embryo fibroblasts, a murine oligodendroglioma cell line, and on MRC5 human fibroblasts and two human glioblastoma cell lines. Class II MHC was much more strongly inhibited (sometimes completely) than class I MHC. TGF also inhibited induction of class I MHC expression by IFN. However, TGF did not inhibit class I or class II MHC induction by IFN in any of the nine colorectal carcinoma cell lines, although two of five of the lines tested were growth-inhibited by TGF. On the other hand, human ICAM-1 induction by IFN was not affected by simultaneous treatment with TGF in any of the cell lines. The down-regulation of IFN-induced MHC antigens by TGF is not, therefore, the result of a general antagonism of IFN. Retinoic acid has recently been reported to induce ICAM-1 expression on human tumour cells. We have confirmed this observation on MRC5, and the two human glioblastoma cell lines, however six colorectal carcinoma cell lines tested did not respond. In contrast to IFN-induced ICAM-1 expression, retinoic-acid-induced ICAM-1 expression was inhibited by TGF on two of the three responsive lines.     

Maintenance of the activation of peritoneal macrophages in patients with ovarian cancer by sizofiran and recombinant interferon-γ

Four patients with ovarian cancer received 20 mg of sizofiran, a -1,3-glucan (molecular weight: 450,000), intramuscularly one day before and 4, 7, 11, 14, 18 and 21 days after second look laparotomy and recombinant interferon- (rIFN-) intraperitoneally on the day of second look laparotomy and 4, 7, 11, 14, 18 and 21 days thereafter.The peritoneal cavity was washed with physiological saline and peritoneal macrophages (Mø) were isolated. The number of Mø increased 30-1600 times during the treatment period. The concentrations of interleukin-1, interferon-, tumor necrosis factor and prostaglandin E₂ were also found increased in the supernatant fluid of Mø cultured for 24 hours with 10 µg/ml of lipopolysaccharide. The present study demonstrated that the activation of peritoneal Mø could be maintained and its number was increased by repeated dosing of sizofiran and rIFN- in combination every three or four days in patients with ovarian cancer. Peritoneal Mø thus activated may exert an antitumor effect on ovarian cancer.     

IL-10 and IFN-γ gene expression in chronic Chagas disease patients after in vitro stimulation with recombinant antigens of Trypanosoma cruzi

Along with several other aspects of Chagas disease, the mechanisms responsible for the different clinical outcomes observed in chronic infected individuals have not yet been clarified. It is believed that the host immune response to the parasite plays an important role in the development of the pathology. Therefore, the aim of this study was to evaluate the relationship between IL-10 and IFN-γ gene expression profile, after in vitro stimulation of peripheral blood mononuclear cells (PBMC) with Trypanosoma cruzi recombinant antigens CRA (cytoplasmatic repetitive antigen) and FRA (flagellar repetitive antigen), and the clinical forms of chronic Chagas disease. Twenty patients with the cardiac form of the disease (CARD), of whom 10 had the mild cardiac form (CARD 1) and 10 the severe cardiac form (CARD 2), and 20 patients with the indeterminate form (IND), were selected at the Chagas Disease Unit of the Oswaldo Cruz University Hospital, University of Pernambuco, Recife, Pernambuco, Brazil. The PBMCs of these individuals were cultured in the presence of CRA or FRA for 3 days and IL-10 and IFN-γ gene expression was evaluated by detection of its messenger RNA using Real Time Quantitative PCR. Although no significant difference was observed between the groups of individuals studied, we found that most patients with IND displayed high levels of IFN-γ gene expression, while the majority of patients with CARD 1 presented high levels of IL-10. The results of this study thus highlight the important role that inflammatory cytokines play in patients with the IND group controlling for parasite replication, and that anti-inflammatory cytokines play in determining susceptibility to progression to symptomatic clinical forms of the disease.     

Loci controlling lymphocyte production of interferon γ after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1–Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2 ^pz ) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2 ^b ) or BALB/cHeA (H2 ^d ) mice, or by ConA. IFNγ production in MLCs of individual (O20 × OcB-9)F₂ mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.     

Clonal analysis of peripheral blood lymphocytes from three patients with advanced neuroblastoma receiving recombinant interleukin-2 and interferon α

In this study we have investigated, at the population and the clonal levels, the immunophenotypes and the non-specific cytotoxic functions of peripheral blood lymphocytes from three stage IV neuroblastoma patients receiving treatment with recombinant interleukin-2 (IL-2) and interferon (IFN). Both IL-2 alone and the combination of IL-2 and IFN caused an in vivo expansion of CD56⁺, CD3^– NK cells most of which expressed the p75 molecule, i.e. the chain of the IL-2 receptor. Peripheral blood mononuclear cells (PBMC), drawn after treatment, displayed an increased NK activity, but no lymphokine-activated killer (LAK) activity. However, the subsequent in vitro culture of PBMC with high-dose IL-2 induced the generation of a potent LAK activity, which was mediated by an expanded population of CD3⁺ CD8⁺ T cells. Finally lymphocytes that had been isolated after cytokine therapy were cloned, in the presence of low-dose phytohemagglutin, immediately or following culture with IL-2. Clones derived from LAK cells expanded in vitro had predominantly a CD3⁺, CD8⁺ immunophenotype, whereas those raised from freshly separated lymphocytes were either CD3⁺, CD4⁺ or CD3⁺, CD8⁺ in equal proportions. Most of the above clones were poorly or not at all cytolytic against NK-sensitive or NK-resistant targets. In contrast, the few NK clones obtained (CD3^–, CD56⁺) lysed all targets with high efficiency.This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro, Milano, Italy to V. P.