Human leukocytic pyrogen, a monokine produced by stimulated human mononuclear phagocytes, will enhance the murine thymocyte proliferation response to phytohemagglutinin (lymphocyte activating factor (LAF) activity). During all steps of purification of human LP, pyrogenicity and LAF activity are coincidental suggesting a single identity for the two monokines. The LAF assay for human LP is highly sensitive and can detect human LP at a concentration of 10−12M. Further experiments suggests that human LP and LAF activities could be destroyed by heating to 70 °C. Furthermore, while in vivo pyrogenicity of human LP can be blocked by ibuprofen, the in vitro LAF activity of the same molecule is unaffected by ibuprofen. Immune rabbit serum directed against human LP could also block in vitro LAF activity either by preincubation with LP or by blocking during culture.
Publisher Summary This chapter examines the role of lymphotoxins in natural cytotoxicity. Lymphotoxin are cytostatic or cytotoxic glycoproeins produced in vitro by activated lymphocytes. In a study described in the chapter, human peripheral blood lymphocytes (PBL) were obtained from defibrinated peripheral blood by the Ficoll–Hypaque technique. The PBL were then cultured at a cell density of 2 × 10 6 cells/ml in RPMI-1640 medium supplemented with 10% FCS (RPMI-10%) at 37°C for 5 to 7 days. At the end of the 5 to 7 days of culture, the cPBL were then used in either a direct cell killing assay or restimulated with 10 μg of Con A/ml at a cell density of 2 × 10 6 cells/ml in fresh RPMI-10% for 5 h at 37°C. The effector cells generated by this method caused significant lysis of the NK-sensitive target cells K-562 and MOLT-4F and little or no significant lysis of the continuous B cell lines Jy or RPMI-1788.
This chapter examines the natural killer (NK) activity in human ovarian tumors. The defective NK activity of tumor-associated lymphoid cells (TAL) could, in principle, be accounted for by at least two mechanisms, one being the presence of suppressor cells or factors and the other being a low number of the relevant effectors, identified in blood as large granular lymphocytes (LGL), which enter the tumor. In a study described in the chapter, preliminary evidence for the presence in situ of cells inhibiting NK activity was obtained in 3 peritoneal effusions from ovarian cancer in 2 breast carcinomas and in pleural carcinomatous effusions. A relatively large number of ovarian cancer patients are examined for the presence of suppressor TAL or TAM. In part of the experiments, TAL or tumor-associated macrophages (TAM) were mixed in varying ratios with PBL and lysis of K562 cells was measured immediately, whereas in others, suppressors were allowed to interact with PBL for 20 h before adding indicator PBL.TAM were devoid of suppressive activity on NK cells when the assay was performed immediately after mixing, whereas in a minority of TAL preparations, the preliminary indications for the presence of inhibitory cells at the tumor site were confirmed.
This chapter describes the natural killing of hematopoietic cells. The major emphasis on natural killer (NK) cells has been their role as an immunosurveillance mechanism against tumor growth. NK cells may have a more physiological role as regulators of normal hematopoietic cells. This hypothesis is based on the findings that mouse NK cells can lyse BM cells and a subpopulation of immature thymocytes from young mice, together with the findings that the in vivo mechanism of bone marrow graft resistance shares several characteristics with the NK system. Data from both mouse and human tumor target systems have shown that when certain tumors are induced to differentiate into more mature stages, they become less sensitive or resistant to NK lysis. The interpretation of the consistent difference in sensitivity between fetal and adult BM is that the stage of differentiation is of importance also in NK lysis of normal targets. The monoclonal antibody OKT 10 is believed to define an early stage of prethymocytes in the BM and is a common marker for all thymocytes.
Publisher Summary This chapter reviews studies on the regulation of tumor growth by natural killer (NK) cells in nude mice. Repeated injection of anti-asialo GM1 (anti-GAl) every three days into mice maintain the NK activity at less than 5% level and abrogated GA1+ cells in the spleen without elimination of T and B cells. It was judged adequate for study of the biological role of NK cells in vivo. Two million syngeneic tumor cells, RL♂-1 or two million allogeneic tumor cells, YAC-1, were subcutaneously injected into each BALB/c nude mouse on the same day, and each mouse also received intravenous injection of rabbit anti-GAI or normal rabbit serum (NRS). For a duration of 24 days, each mouse received repeated injections of the antiserum every three days and the size of palpable tumors was recorded. 100 percent palpable tumor of RL♂-1 appeared in anti-GAl injected mice, whereas the tumors were detectable less than 25% of mice injected with normal rabbit serum (NRS).
Publisher Summary This chapter explores the natural killer (NK) activity of tumor infiltrating and lymph node lymphocytes in human pulmonary tumors. In a study described in the chapter, 53Cr-release assays against K562 targets showed low activity among tumor infiltrating lymphocytes (TIL) from pulmonary tumors of a variety of histologic types. The TIL was obtained by mechanical disaggregation, fractionated by filtration through nylon monofilament mesh to remove tumor cells. The removal of residual tumor cells did not result in improvement of NK activity, indicating that the low NK activity is not because of cold target competition by residual tumor cells. TIL obtained from tumors injected two weeks prior to surgery with intralesional BCG exhibited higher levels of NK activity than TIL from uninjected tumors. Similarly, hilar lymph node lymphocytes and peripheral blood lymphocytes from the same patients showed modestly higher NK activity. In three cases, BCG-injected TIL and uninjected TIL from the same patients. These patients had multiple pulmonary metastases, only one of which was BCG injected. It was found that in these cases, the NK activity of uninfected TIL remained low while the NK activity of TIL from the adjacent BCG-injected tumors were high.
This chapter focuses on one type of natural killer (NK)-activity that could be called classical with the YAC-1 lymphoma as the prototype target cell. Importance of the D-end of the H-2 complex against YAC-1 in a larger series of B10 congenie strains has been confirmed. In a study described in the chapter, assays of NK cell activity were performed with normal spleen cells of individual mice. B6 mice were included in each test to provide a reference as the sensitivity of the assay varied considerably. It is, in fact, possible that the gene is identical to the gene coding for the H-2 D or L molecule. The other gene regulating NK-activity is telomeric of H-2 D but centromeric of Qa-2 and, thus, defines a new locus, NK-activity gene (NKT).
Natural resistance to infection with several intracellular pathogens— both bacterial and protozoal—has recently been demonstrated to be under genetic control. The ability of genetically-resistant mouse strains to prevent growth, in the reticuloendothelial tissues, of Mycobacterium bovis (BCG) is controlled by a single, dominant, autosomal gene designated Bcg. In a study described in the chapter, mice of 17 inbred strains were infected intravenously with 104 CFU of M. bovis (BCG) and Bcg typed 3 weeks later as either resistant (r) or susceptible (s) according to the level of bacterial burden in their spleens. Because the strain survey and the analysis of recombinant inbred strains suggested a close linkage (or identity) of Bcg, Lsh, and Ity genes, a formal proof was sought by the examination of individual animals obtained from a segregating population for the distribution of the three phenotypes in question. Twenty-six backcross animals and the appropriate parental and F1 hybrid controls were infected with BCG and they were splenectomized three weeks later for Bcg typing. The recent availability of mouse strains that are congenie except for the Bcg (Lsh, Ity) gene should greatly enhance the functional studies on the phenotypic expression of this gene.
This chapter examines the modification of human natural cell-mediated cytotoxicity by maleic anhydride and divinyl ether (MVE-2). In a study described in the chapter, a total of 21 evaluable patients with advanced refractory neoplastic disease were entered into the Phase I study of MVE-2. MVE-2 at the appropriate dose level was infused intravenously over two hours at weekly intervals for a total of six weeks. Three patients were entered at 150 mg/m2, four at 300 mg/m2, four at 350 mg/m2, one at 400 mg/m2, four at 450 mg/m2, and five at 600 mg/m2. Patients were monitored for biological response modification by measuring 29°C E-rosettes, 4°C E-rosettes, phytohemagglutinin (PHA)-induced blast transformation, pokeweed mitogen (PWM)–induced blast transformation, natural cytotoxicty to K-562, plasma interferon levels, and plasma lysozyme levels in the peripheral blood. All tests on cells were performed on Ficoll–Hypaque separated cells. All assays were performed twice prior to administration of MVE-2 and weekly thereafter for as long as the patient remained in the study. No significant changes in the levels of response to the T-cell mitogen PHA were noted during MVE-2 administration. However, nine of eighteen patients tested showed increased responses to pokeweed mitogen.
This chapter describes the Beige model in studies of natural resistance to syngeneic semisyngeneic and primary tumors. The concept of natural surveillance against tumors postulates that the immune system can recognize and eliminate neoplastic cells before they give rise to overt malignancies. The role of natural killer (NK) cells in this hypothetical defence is sometimes discussed in terms of spontaneous tumor incidence in various inbred strains of mice that show constant differences in their levels of NK activity. The chapter presents two different models where the effect of the bg mutation on natural resistance can be studied in mice with thymus deficiency. In the first approach, beige-nude mice were obtained through a breeding scheme. The bg mutation was found to cause a defect in NK function also in nude mice, although the homozygosity for the nu alleles had compensated partly for this by elevating the NK activity above the very low levels observed in beige mice without the nu defect. The data indicate that the mechanism responsible for resistance in +/bg mice which is defective in bg/bg mice is bone-marrow derived and does not require an intact thymus to develop.
This chapter focuses on characterization of effector populations mediating natural killer (NK), natural cytotoxic (NC), antibody-dependent cellular cytotoxicity (ADCC), and spontaneous macrophage cytotoxicity in murine peritoneal exudate cell preparations. The studies described address two questions: (1) are the lysis of the NK susceptible YAC-1 and the NC-susceptible Meth-A tumor cell lines the function of the same population that manifests ADCC and SK killing and (2) is it possible to separate or enrich the various populations and thus facilitate their characterization. In a study described in the chapter, resident noninduced peritoneal cells were obtained from 8–12 week (C57BL/6 × DBA/2)F1 mice by lavage. Viable nucleated cells were suspended in mouse tonicity phosphate buffered saline (320 m0sm) and separated by velocity sedimentation at unit gravity according to the method of Miller and Phillips. After sedimentation, 35 ml fractions were collected, washed, counted, and appropriately diluted. NK, NC, and ADCC cell functions were tested. Significant enrichment and separation of cytotoxic effector populations can be achieved using velocity sedimentation. Velocity sedimentation may represent a valuable pre-enrichment method for secondary characterization studies.
This chapter examines induction of natural killer (NK) cells and interferon production during infection of mice with Babesia Microti (B. microti) of human origin. To examine the effect of B. microti infection on NK activity, in a study described in the chapter, groups of female +/+, +/bg, and bg/bg mice were infected intravenous with 108 parasites in +/+ blood. Peak induced NK cytotoxicity occurred on day 2 postinfection in each mouse strain, at levels that were comparable to those observed after inoculation of mice with NK-inducing agents such as poly I:C or VSV. Similar results were obtained when the size of the parasite inoculum was reduced. On days 4, 6, 8, and 21 postinfection, spontaneous cytotoxicity in B. microti-infected mice decreased toward or below control values. The level of NK activity of the infected bg/bg mice on days 1, 2, and 4 was approximately equal to the normal level of NK activity observed in wild-type mice. Thus, the relative increases in NK activity because of B. microti infection were similar in beige and wild-type mice, although the absolute levels of NK activity in the two mouse strains were always significantly different.
This chapter examines in vivo effects of biological response modifiers and chemotherapeutic agents on natural killer (NK) activity in cancer patients. The study described in the chapter investigated the possibility that NK cell activity may be of value as an effective screening test for new drugs and for a precise evaluation of the immune status of the cancer patient. In vivo testing was evaluated in immunodeficient, advanced solid tumor patients. Blood samples were drawn on the morning of the first day and on the day following cessation of treatment. Patients were tested for NK activity. NK activity was measured by a chromium 51 assay. K562 target cells were marked with chromium-51 and placed in the wells of a linbro round-bottom microtest plate with different concentrations of human lymphocytes, which were separated from peripheral blood by the Boyum technique using Ficol–Metrizoate (Pharmacia) at 1.077 g/cm3. The cells were incubated for 4 h at 37°C in a humid CO2 incubator. Results demonstrated that there was a strong increase in NK activity in patients treated by human fibroblastic interferon, bestatin or cis-platinum. Only a slight increase was noted for immunodeficient patients treated by retinoic acid RO 109359.
This chapter describes the target cell recognition by natural killer (NK) and natural cytotoxic cells. Natural cell-mediated cytotoxicity (NCMC) is the function of at least two distinct effector cell populations, NK, and natural cytotoxic cells. Studies from a number of laboratories have resulted in significant characterization of the effector populations; however, little is known regarding the nature and specificity of their respective target cell recognition structures. Cross cold target inhibition studies have been valuable in comparing a number of tumor cell lines for the presence or absence of such antigens. The inhibition of NK and NC cell activities by monosaccharides has led us to suggest that lectin-like receptors may be involved in the effector–target interaction. On the other hand, the isolation of a resistant variant of the NK susceptible L-5178 lymphoma has resulted in the association of the neutral glycolipid asialo-GM2 with NK-susceptibility. Antigens encoded by the major histocompatibility complex are involved in cell–cell interactions leading to the generation and function of cytotoxic T lymphocytes. The studies show that variant tumors with differences in susceptibility to NCMC represent possible models for the definition of NCMC target antigens.
Publisher Summary This chapter describes the recognition structures for natural killer (NK) cells on human lymphocytes. Using lymphoblastoid cell lines as target, pretreatment of effector cells inhibited or decreased the NK-activity. Similarly ADCC, against the same target cells sensitized by specific HLA antibodies, was also inhibited. The cytotoxic effect on autologous PHA blasts of CTL and autologous and allogeneic effect of NK-cells generated in a 6 day old mixed lymphocyte culture was not affected by anti-BMi, although an inhibitory effect was observed on Daudi target cells. Using a panel of 29 autologous and allogeneic PHA target cells and measuring the cytotoxic activity of Ncp effector cells of the same donors, distinct cytotoxic responses were recorded in the short-term assay. The highest cytotoxicity was 56%, and spontaneous release was under 17%. Target-cell sensitivity and effector cell reactivity varied four times and seven times within the respective maximum values. The chapter presents selective cytotoxycity scores in autologous and allogeneic combinations. Autologous reactivity was nil or very low /up to ±4 /in 21 donors who are, thus, regarded as nonreactive persons.
This chapter analyzes differentiation events causing changes in natural killer (NK) cell tumor–target sensitivity. The chapter presents data obtained in the analysis of freshly isolated human chronic lymphocytic leukemia (CCL) cells and a murine myeloid leukemia cell line undergoing induced controlled differentiation in vitro. The study described in the chapter analyzed the way in which the induction to differentiation altered the NK sensitivity of the tumor cell population and its ability to be lysed by homologous or xenogeneic effector cells and whether the alteration in sensitivity affected the competing capacity of such cells when known NK target cells were used. Freshly obtained tumor cells from B-CLL patients can, in certain cases, be induced to differentiate in vitro, if treated with the phorbol ester 12-0-tetradecanoyl-13-phorbolacetate (TPA). The total material included 18 B-CLL patients tested at 35 different occasions during a time period of 18 months. A substantial increase in NK cell susceptibility was noted in 19 cases (responders). This increase peaked at the third day of culture. No change in background sensitivity was shown by the cells cultured in the absence of TPA.
This chapter describes the heterogeneity of mixed lymphocyte culture-generated natural killer (NK)-like cells. Allogeneic MLC has been shown to generate NK-like cells in addition to CTL. This in vitro generation of NK-like activity allows the characterization of NK-like effectors, tracing of their development and maturation, and examination of the cytotoxic specificity of these cells. A MLC provides an excellent model to compare the development and killing behavior of CTL and NK-like cells. MLC-generated cells were tested daily for 7 days in the single cell assay in which both targets were used together. The alloantigen target cells were trypsinized to prevent their binding to other cells and also fluoresceinated to distinguish them from the MLC-generated effectors. During the 7 days of MLC, no simultaneous binding of both targets was seen. This assay can detect cells capable of simultaneously binding two targets. If NK is a function of the developing alloreactive lymphocyte, there should be a point in time when a cell changes from NK reactivity to alloreactivity.
Exponential proliferation of T-cell lines developed from mitogen-stimulated T-lymphocyte clonal expansion has been found to be highly dependent upon the presence of T-cell growth factor, designated as Interleukin-2 (IL-2). Initiation of clonal expansion has been shown to be positively regulated by Interleukin-1 (IL-1) and negatively regulated by prostaglandin E2 (PGE2), which are both produced by monocyte/macrophages. The T-cell colonies used in these experiments described in this chapter were obtained from peripheral blood mononuclear cells from healthy donors. Individual T-lymphocyte colonies were removed from soft agar culture and then placed into liquid medium containing 1.0 unit per ml IL-2 until a density of 7.0 × 105 lymphocytes per ml or greater was obtained. The presence of IL-2 during clonal proliferation may have a critical impact on functional expression and T-cell colony selection. The results indicate that IL-2 causes significant augmentation of NK activity in 18 h incubation. The effect of IL-2 in the system indicates that under standard conditions of T-cell colony establishment, expression of natural cytotoxicity would be strongly promoted.
This chapter discusses the different aspects of ethanol and natural killer activity. In a study described in the chapter, natural killer activity of human peripheral blood lymphocytes (HPBL) was studied in a 4 hr 51Cr release assay using K562 target cells. HPBL preparations derived from a total of 15 normal healthy volunteers and 32 chronic alcoholics were examined for NK activity in 11 experiments. In each experiment, at least one control HPBL preparation was tested along with two to six HPBL preparations from alcoholics. Wide variations in the levels of NK activity were found in HPBL preparations from control and alcoholics, but upon statistical analysis of variance, levels of NK activity in PBL derived from alcoholics were significantly higher than the control levels. The difference in the cytotoxic activity of PBL from control and alcoholic subjects was not abolished as a result of depletion of macrophages, T cells, and B cells, and therefore the cytotoxic activity of PBL from normal and alcoholic subjects resided in the NK population. The NK inhibitory effect of ethanol could be confirmed in the mouse system. Short-term preincubation of mouse spleen cells with ethanol had no effect on their subsequent NK activity.
