Eva Lotzová

Inhibition of clonogenic growth of fresh leukemia cells by unstimulated and IL-2 stimulated NK cells of normal donors

We have demonstrated that unstimulated highly-enriched NK cells have the capability to inhibit the growth of fresh clonogenic leukemic cells from AML, CML and preleukemic patients. The NK-cell population mediating antileukemic reactivity exhibited LGL morphology and NKH1 and CD16 phenotype. The inhibition of leukemic growth could be mediated by cell-to-cell contact or by soluble factor produced by NK cells. Antileukemia activity was only detectable when enriched population of LGL was utilized; NW-filtered lymphocyte population did not exhibit leukemia-inhibitory effect. However, such activity could be generated after culture of the latter effector cells with IL-2. The leukemia directed IL-2 activated effector cells were characterized as NK cells. The data reported here provide new insight into host factors which may control leukemia growth and indicate the possible future application of NK cells for therapy of leukemia.

Natural killer cells in mice and man and their possible biological significance

ConclusionsDespite active and extensive research on human and murine NK cells in the last few years a number of basic questions remain to be answered. These include the nature of the antigen(s) on target cells to which NK cells react, the nature of the receptors on NK cells allowing the recognition, the mechanism of NK cell cytotoxicity, the exact relationship between NK cell activities in vivo and in vitro, genetic control of NK cells, involvement of NK cells in the field of bone marrow transplantation, and finally their involvement in immunosurveillance of malignancies. Although the etiology and biologic significance of NK cells remain obscure, there is suggestive evidence that these cells may be in volved in resistance to and immunosurveillance of malignancies, since there is experimental evidence for a correlation between in vivo and in vitro NK cell activities. The fact that athymic mice do not have a higher incidence of malignancies than conventional mice and express delayed appearance and higher resistance to virally and chemically induced tumors (Stutman, 1975) is consistent with a protective role of the NK cell effector mechanism. In addition, their spontaneous occurrence without any necessity for priming and their ability to recognize and kill malignant cells makes NK cells almost perfect candidates for the part of immunosurveillors. The similarity between bone marrow effector cells and NK cells also seems unlikely to be fortuitous. In summary, this interesting cell requires further attention.

Natural immunity against ovarian tumors.

We have analysed natural killer (NK) cytotoxic activity in peripheral blood and ascitic fluids of patients with advanced stage of ovarian epithelial carcinoma. All patients displayed low NK activity in peripheral blood and virtually no cytotoxicity in ascitic fluids. NK activity in ascitic fluids could be substantially augmented after regional administration of virus-modified tumor cell extracts (VMTE), and that in peripheral blood after culture of effector cells with interleukin-2 (IL-2) in vitro. Activated NK cells displayed cytotoxic activity against NK-sensitive and NK-resistant tumor cell lines as well as against fresh ovarian tumors. Parallelism was found between regional NK augmentation and regression of malignant ascites. The latter observation suggests possible NK cell role in defense against ovarian tumors.

Highly oncolytic adherent lymphocytes: Therapeutic relevance for leukemia

We have generated and characterized a highly oncolytic adherent lymphocyte subset (A-LAK) from eight leukemic patients with non-lymphocytic leukemia (NLL) in remission and one NLL patient in relapse. Our studies demonstrated that A-LAK was superior in its oncolytic activity (tested in a 3-h 51Cr release assay) to conventionally prepared (LAK) and non-adherent (NA) IL-2 cultures. No activity was observed by this highly oncolytic subset against normal bone marrow (BM). A-LAK also displayed highest proliferative activity in 7-11 day cultures (5- to 58-fold expansion) in comparison to LAK (0.7- to 2.7-fold) or NA (1.0- to 2.6-fold) cultures. Analysis of phenotype of unseparated, NA and adherent (A-LAK) lymphocytes 24 h after IL-2 activation showed that the A-LAK was composed predominantly of high intensity (bright) CD11a+ (LFA-1) lymphocytes (75 +/- 4.8%) when compared to the other two populations (12 +/- 2.1%). Similarly, A-LAK contained higher proportion of CD11b (CR3 receptor)-positive lymphocytes (39 +/- 2.1%) than unseparated and NA lymphocytes (11 +/- 1.4%). Double marker phenotypic studies showed that A-LAK cultures were heterogeneous and distribution of individual lymphocyte subsets differed among NLL patients. While in A-LAK culture of some patients the CD56+, CD3- natural killer (NK) cell subset was predominant, CD3+, CD56- lymphocyte subset was prevalent in others. Highest A-LAK lytic activity was always correlated with highest NK cell content. Characterization studies (using the complement-depletion technique) showed that independently of the distribution of lymphocytes in A-LAK cultures, CD16+, CD56+, CD3- NK cell subset displayed highest oncolytic effect. CD5+ subset also participated in cytotoxic function. These observations indicated that A-LAK may represent a new therapeutic approach to treatment of leukemia.

Inhibition of murine natural killer cell-mediated cytotoxicity by pretreatment with ammonium chloride

In the present study the effect of ammonium chloride on murine natural killer (NK) cell-mediated cytotoxicity to T cell lymphoma, YAC-1 was studied. It was found that ammonium chloride treatment significantly reduced the cytotoxicity of splenic NK cells without any detectable change in cell viability. It is, therefore, suggested to avoid ammonium chloride treatment in order to obtain the realistic reflection of murine NK cell activities.

Interleukin-2 corrects defective NK activity of patients with leukemia

NK cells of patients with leukemia display low cytotoxic potential. Since the NK cells have been suggested to play a role in natural resistance to leukemia, we considered it of importance to investigate the approaches leading to the correction of NK defect of leukemic patients. Our studies demonstrate that culture of effector cells with interleukin-2 (IL-2) resulted in restoration of cytotoxic defect. This was indicated by normalization of tumor-binding as well as lytic NK activity, by normal frequency of cytotoxic cells and their ability to recycle. The NK cell nature of cytotoxic cells was shown by abrogation or depletion of cytotoxicity by antibody directed against NK cell-associated, but not T cell-associated antigen. The generation of NK cell activity against fresh leukemic cells suggests that adoptive transfer of IL-2 activated NK cells may be a new approach to leukemic treatment.

Pyrimidinones: Inducers of NK cell activity and antitumor immunity

We have shown that pyrimidinone molecule, 2 amino-5-iodo-6-phenyl-4-pyrimidinone (AIPP) after regional administration, displayed both prophylactic and therapeutic effect on ascitic mammary adenocarcinoma, ACA-755. The antitumor effect was mediated by natural killer (NK) cells, since depletion of this lymphocyte population resulted in abrogation of AIPP-induced tumor resistance. This observation confirms the role of NK cells in antitumor immunity and suggests that AIPP may be therapeutically beneficial also in man.

Studies on the antitumor activities of pyrimidinone-interferon inducers. Part 2 Potentiation of antitumor resistance mechanisms

In continuation of studies on antitumor activities of pyrimidinone interferon inducers [11], we report here that 2-amino-5-bromo-6-mF-phenyl-4(3H)-pyrimidinone (ABmFPP) is similarly effective to 2-amino-5-bromo-6-phenyl-4-pyrimidinone (ABPP) in its ability to reduce the number of metastatic nodules of a spontaneous fibrosarcoma (NFSa) and a spontaneous mammary carcinoma (MCa-K) in the lungs of C3Hf/Kam mice. Both compounds were more effective when given to mice prior to, rather than after, intravenous transplantation of tumor cells. In studies on the mechanism of the antitumor activity of pyrimidinones, 2-amino-5-iodo-6-phenyl-4-pyrimidinone (AIPP) was used in addition to ABPP and ABmFPP. These agents were capable of activating peritoneal macrophages that thus became capable of lysingin vitro 3T12 transformed cells but not syngeneic BALB/c embryo fibroblasts. Also, these agents were capable of augmenting significantly the natural killer (NK) cell activity in the spleen of C3Hf/Kam mice. Spleen cells from treated mice admixed to NFSa cells inhibitedin vivo tumor take of these cells when the admixture was injected subcutaneously. Pyrimidinones were also effective against the development of NFSa nodules in the lungs of T-cell deficient mice implying that the presence of T-cells is not a prerequisite for the induction of antitumor activity by these agents. A further observation was that pyrimidinone compounds reduced the metastasis formation enhancing effect of cyclophosphamide. Therefore, pyrimidinone interferon inducers exhibit an appreciable antimetastatic activity mediated through antitumor resistance mechanisms involving activation of macrophages and stimulation of NK-cells.