Howard T. Holden

Natural Cell-Mediated Immunity

Publisher Summary The recognition of the existence of natural cell-mediated immunity, particularly natural cell-mediated cytotoxicity, has significantly altered the concepts concerning the potential mechanisms for in vivo resistance against tumor growth and for in vitro cell-mediated immune reactions. When cytotoxic reactions are measured, the evaluation of the role of natural killer (NK) cells and other more well-known mechanisms of cytotoxicity must be done carefully. This chapter focuses on natural cell-mediated cytotoxicity. It summarizes the known information on the expression of natural cytotoxicity in rodents and in man, its specificity, the nature of the effector cells and their relationship to other immune mechanisms, and the possible in vivo relevance of natural cytotoxicity, particularly in regard to resistance against tumor growth. The basic observation that initiated the studies of natural cell mediated cytotoxicity includes that lymphoid cells from some normal mice, rats, and human donors, which are not inoculated with tumor cells or other sources of antigen, exhibit significant levels of cytotoxic reactivity against certain syngeneic or allogeneic tumor cells.

Natural killer cells. Characteristics and regulation of activity.

Recently there has been increasing recognition of natural cell-mediated cytotoxicity as a potentially important antitumor effector mechanism in addition to that of specifically immune T cells and of activated macrophages. Although natural cellular cytotoxicity was first recognized only a few years ago (Herberman et al. 1973,1974, McCoy et al. 1973b, Oldham et al. 1973, Rosenberg et al. 1972,1974), there has already been extensive research in many laboratories on the nature of the effector cells, the possible mechanisms of cytotoxicity, the factors regulating the levels of reactivity, and the relevance of natural immunity to in vivo resistance against tumor growth and immune surveillance. A principal component of natural cell-mediated cytotoxicity in rodents and man has been found to be a particular subpopulation of lymphocytes which have been termed natural killer (NK) cells. We have recently reviewed in detail much of the available information on NK cells (Herberman & Holden 1978). In this paper we will only summarize our views on the characteristics of NK cells and focus on a few issues of current interest in our laboratory.

Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide.

Abstract Macrophages derived from MSV-induced tumors and several macrophage cell lines showed direct cytolytic activity in an 18-hr 51 Cr release assay against tumor target cells. The cytolytic activity of these macrophages was augmented by the addition of muramyl dipeptide (MDP) to the cytotoxicity assay, an effect similar to that observed with bacterial lipopolysaccharide. The stimulation of macrophage-mediated cytotoxicity by MDP appeared to be under genetic control since macrophages from BALB/c mice were augmented with MDP while those from C57BL/6 animals were not. MDP appears to act directly on the macrophage without the participation of any other cell type, since MDP increased the activity of the macrophage cell lines.

Measurement of macrophage-mediated cytotoxicity against adherent and non-adherent target cells by release of 111indium-oxine

This report describes the utilization of 111 indium-oxine chelate ([111In]Ox) for studies of macrophage-mediated cytotoxicity. [111In]Ox efficiently labeled both non-adherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intracellularly incorporated [111In]Ox was very slow (0.25-0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [111In]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [111In]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [111In]Ox released in response to cytolytic macrophages correlated well with those observed for the 51Cr and [3H]TdR radiolabels. Therefore, [111In]Ox can be utilized for relatively short-term (less than 20h) assays with lymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity.

Immunologic Reactivity of Lymphoid Cells in Tumors

There have been many studies of immune responses against tumors and almost all of these have focused on the reactivity in the blood or spleen. From such studies, it has become clear that a wide variety of effector cells and types of immune functions may be involved in antitumor responses. Particular attention has been directed toward T cells that may be directly cytotoxic against tumor cells or may proliferate or produce lymphokines upon stimulation with tumor antigens. However, other effector mechanisms may be involved and need to be considered. These include B cells, which can produce antibodies that affect tumor cells directly or that interact with K cells or macrophages and thereby mediate antibody-dependent cell-mediated cytotoxicity; macrophages and monocytes, which are spontaneously cytotoxic or can be activated to become cytotoxic against tumor cells; and natural killer (NK) cells, a subpopulation of lymphocytes with spontaneous cytotoxic reactivity against tumor cells.

Endotoxin requirement for macrophage activation by lymphokines in a rapid microcytotoxicity assay

A short term microcytotoxicity assay is described for studying the activation of M0 by lymphokines. Proteose-peptone-induced macrophages were purified by adherence in flat-bottomed microtiter plates and then activated by MAF-containing supernatants for 18 h. 51Cr-labeled tumor target cells were added for an additional 18 h, and then the supernatants were harvested and the % isotope release quantitated. When endotoxin-free medium and FBS were used, we found that small amounts of LPS were absolutely required for macrophage activation in this assay. The advantages of this technique included (a) good reproducibility, (b) the requirement for small number of M0, and (c) the potential of standardizing the assay and thereby testing a large number of samples. Moreover, this assay may have particular value for investigations of M0 activation by exogenous stimuli, since M0 that were not pretreated with activating agents did not exhibit cytotoxicity.

Suppression of lymphokine production. I. Macrophage-mediated inhibition of MIF production

Abstract Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum , whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.

Immune response to gross virus-induced lymphoma cryopreservation of functional activity of rat lymphocytes and tumor cells

Abstract We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr 51 Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., 3 H-proline and 125 IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A) 3 or tumor cells (MLTI) as measured by 3 H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity. By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.

IMMUNOGENICITY OF TUMOR ANTIGENS

A central theme in experimental tumor immunology is the immunization with tumor antigens. The objectives of the studies on this subject have usually been the induction of either transplantation protection against tumor challenge or of in vitro immune reactivity. Few detailed studies have been performed to determine mechanisms for in vivo immunogenicity, by relating those data to results obtained in vitro. In this paper, we will describe a series of experiments performed primarily with virus-induced tumors of mice and rats, in which some of the factors that may influence in vivo immunogenicity are analyzed. It has become clear from these studies that some “nonimmunogenic” tumors or tumor cell preparations are antigenic and under some circumstances can induce an immune response in the host. We have placed particular emphasis on lymphoproliferative responses and on cell-mediated cytotoxic reactions, after primary or secondary exposure to tumor antigens in vivo or in vitro, and these experiments have been quite helpful for the analysis of immunogenicity. A number of factors have been identified that may interfere with immunogenicity of antigenic tumors: Suppressor cells in the host, immunosuppressive viruses in the tumors, and specific and nonspecific serum factors. Careful dissection of factors influencing immunogenicity of tumor antigens should help to provide practical solutions to problems related to immunoprevention and immunotherapy of cancer, which have not been solved by empirical studies.

Differential in vitro modulation of suppressor and antitumor functions of mouse macrophages by lymphokines and/or endotoxin

Peritoneal macrophages of normal mice exhibited natural suppressor activity, as indicated by their ability to inhibit the proliferation of spleen cells in response to stimulation with phytohemagglutinin (PHA) or concanavalin A (Con A). Their suppressor function could be modulated in vitro with a variety of treatment regimens. High-dose lipopolysaccharide (LPS) (LPSH; 10 micrograms/ml) or lymphokines (supernatant from Con A-stimulated spleen cells) plus low-dose LPS (LPSL; 10 ng/ml) caused a reduction in the suppressor activity of adherent peritoneal macrophages. In contrast, these same treatments induced the macrophages to become tumoricidal and cytostatic for tumor cells, indicating a major dissociation between the regulation of suppressor and cytotoxic activities of macrophages. The lack of correlation between these activities was further demonstrated by macrophages that had been activated in vitro by Corynebacterium parvum: these cells expressed high tumoricidal and cytostatic activities, and also strong suppressor activity. The suppressor function could be selectively downregulated by in vitro pretreatment with LPSH.