Natural and Induced Cell-Mediated Cytotoxicity: Effector and Regulatory Mechanisms

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IMMUNOBIOLOGY OF NATURAL KILLER CELLS: MECHANISMS AND REGULATION IN THE MURINE SYSTEM

Outline

Chapter 1: CHARACTERISTICS OF NATURAL CYTOTOXIC CELLS IN MICE

Chapter 2: GENERATION IN VIVO OF MOUSE NATURAL CYTOTOXIC CELLS

Chapter 3: ARE CYTOTOXIC T CELLS RELEVANT IN THE LOCAL DEFENSE AGAINST SOLID TUMORS?

CHARACTERISTICS OF NATURAL CYTOTOXIC CELLS IN MICE

Rolf Kiessling

Publisher Summary

This chapter describes the characteristics of natural cytotoxic cells in mice. Several groups of investigators have shown that tumor cells can be destroyed by effector cells from normal donors who, as far as could be determined, had neither been immunized nor sensitized. These effector cells have been designated as natural killer (NK) cells in analogy with the phenomenon of natural antibodies. NK-cell phenomena have been studied in mice, rats, and man. Almost all studies have utilized the short term ⁵¹Cr release assay. In the case of rats and man, long-term assays, such as the microcytotoxic assays, have been employed. Target cells cultured in vitro are consistently more sensitive to NK lysis than the respective in vivo lines. It is now well established that in the mouse, the NK cell is not a mature T cell as it lacks detectable amounts of theta-antigen and inasmuch as nude mice exhibit high activity. Mouse NK cells lack surface immunoglobulin as well as C3 receptors.

Several groups of investigators have shown that tumor cells can be destroyed by effector cells from normal donors who, as far as could be determined, had neither been immunized nor sensitized. These effector cells have been designated NK, i.e., natural killer cells, in analogy with the phenomenon of natural antibodies. NK-cell phenomena have been studied in mice, rats, and man. Almost all studies have utilized the short term ⁵¹Cr release assay, and most of the experiments to be considered have been performed with this technique. In the case of rats and man, long-term assays, such as the microcytotoxic assays, have been employed.

SPECIFICITY OF MOUSE NK CELLS

It has been concluded by several groups of investigators that mouse NK cells show a reproducible pattern with respect to target cell sensitivity (1–4). T lymphomas constitute the tumor cell type most susceptible to NK lysis, but other tumors of nonhematopoietic origin also express significant lytic sensitivity (1,2). Susceptibility to lysis by NK cells depends on expression of relevant target structures on the various cell lines as verified in competition assays. In these assays the capacity of various cold competitor cells to inhibit isotope release from labeled target cells was assessed. A good correlation between susceptibility to direct lysis and the ability to inhibit lysis was generally found (1,2). NK-cell-mediated lysis was found to function across H-2 (1) or species barriers (5).

Target cells cultured in vitro are consistently more sensitive to NK lysis than the respective in vivo lines (1, 2). Following in vitro explantation of a mouse lymphoma, this increase in lysis sensitivity did not occur until after three weeks of in vitro culture.

A variety of cell surface components have been considered as the target site for the cytolytic activity of NK cells, but no compelling evidence is available to support any of the hypothetical explanations. The most widely supported notion suggested that NK activity is directed against endogenous C-type viral antigens on the target surface (2). However, Becker and Klein (6) found no correlation between the NK susceptibility of Moloney lymphoma sublines that differed markedly in Moloney virus-determined antigen expression. It is also noteworthy that mouse NK cells can kill certain human targets, T-cell lymphomas, in particular (5). Furthermore, human cell lines that were deliberately superinfected with mouse xenotropic C-type virus by passage in nude mice and subsequently tested for NK sensitivity showed no increase or altered sensitivity to mouse NK cells. Thus, the target specificity attacked by the NK cell remains unknown. It is of interest that recent evidence suggests that NK cells can play a role in resistance not only to certain tumor lines, but also to normal bone marrow grafts (7, 8). Accordingly, NK cells could also play a role in the control of hemopoietic differentiation. In that event they probably recognize a cell type associated with other than viral specificities.

IN VITRO CHARACTERISTICS OF MOUSE NK CELLS

It is now well established that in the mouse the NK cell is not a mature T cell (9, 10) as it lacks detectable amounts of theta-antigen and inasmuch as nude mice exhibit high activity. Furthermore, mouse NK cells lack surface immunoglobulin as well as C3 receptors (9, 10). Despite earlier negative findings (10), Herberman et al., (11) have concluded that NK cells have low but detectable amount of Fc receptors. There is general agreement that the NK cell is poorly adherent and nonphagocytic (3, 4, 9, 10). Accordingly, NK cells in mice are neither mature B nor T cells. Reports describing the rat NK cell seem to be in concurrence with those from mouse systems (12, 13). Here too, the killer cell seems to be of non-T origin as measured by a heterologous anti-T serum. Also, rat NK cells lack C3 and Fc receptors, and are poorly adherent and nonphagocytic.

Even this brief summary suffices to conclude that mouse and rat NK cells constitute a cell type clearly distinguishable from previously defined cytotoxic cells.

NONGENETIC FACTORS INFLUENCING NK ACTIVITY

NK activity in the mouse can be influenced by a number of factors as listed in Table 1.

TABLE 1

Influence of Nongenetic and Genetic Factors

First to be mentioned are some nongenetic methods used to manipulate NK activity. The pronounced influence on NK activity of the age of the animal is noteworthy. Fetal liver and spleen cells from new-born mice show little or no activity. In contrast to immune T cells, which in mice mature during the first week of life, the onset of NK activity does not occur until three weeks of age, and peak activity is seen in 6–8 week old mice. Thereafter there was a marked decline of activity, 6–12 months old mice showing very low activity (1, 2). In rats a roughly similar situation has been observed (12, 13).

There are several lines of evidence that NK cells are of major importance for rejection of subcutaneously injected tumor cells (14, 15). NK cells can probably migrate from the spleen or peripheral blood, organs known to contain the highest NK activity, into the local site of tumor growth. Alternatively, they could be recruited from cells already present in various organs. It will be important to ascertain the influence of tumor induction and of immunization with tumor cells on NK activity in peripheral lymphoid organs. Becker and Klein (6) have studied the NK activity in three separate models of tumor-bearing animals, and in all three systems a pronounced suppression of NK activity in the spleen was found.

Herberman et al. (16) have determined the effect of host challenge on NK activity and found that in nude, as well as in normal mice of various ages, reactivity could be augmented by inoculation of tumor cells. This augmented cytotoxicity reached a peak three days after inoculation, and was only seen with tumor cells that bore target structures recognized by NK cells. The authors concluded that, in most respects, this augmentation of NK reactivity was consistent with the stimulation of specific memory cells by in vivo reexposure to antigen.

It has also been established by the same authors as well as by other groups that NK activity in mice could be augmented by other agents, notably allogenic cells, bacteria, and viruses. Wolfe and collaborators (17) demonstrated that viable BCG organisms given i.p. induced in the peritoneal cavity of normal mice a population of cytotoxic cells. These cells appeared within a few days after BCG administration, and had many features in common with NK cells. Also, i.p. administration by heat-killed Corynebacterium parvum elicited a similar phenomenon (Ojo et al., personal communication). Thus, augmented NK activity after administration of bacterial adjuvants or viruses show that NK cells can be boosted nonspecifically, but the mechanism responsible for this nonspecific boosting is not known.

GENETIC REGULATION OF NK ACTIVITY

Early in the course of the study of NK cells in mice, it was found in different laboratories that various mouse strains differed consistently in NK reactivity (1, 2). This pronounced strain difference is indicative of a genetic influence and was investigated by further genetic analyses. High reactivity appears to be dominant; when the low reactive A-strain was crossed with various other strains, and cells from these F1 hybrids tested for NK activity against semisyngeneic YAC lymphoma of A origin, reactivity resembled that of the high-reactive parent (18). This high NK activity in F1 mice has suggested a possible relationship between NK cells and the so-called hybrid effect. Since the time when Snell (19) first described this phenomenon, it has been shown in several tumor host systems that F1 hybrid animals are generally more resistant to tumor growth of transplanted parental tumor cells than the strain of tumor origin. The precise mechanism operative in F1 hybrid resistance remains obscure. Could NK cells represent this protective mechanism? Support for this notion derives from the fact that both phenomena are influenced by factors in the H-2 complex. The role of H-2 linked factors in the NK system was first shown in the YAC tumor system (20). For this purpose a linkage study in a backcross between the low-reactive A strain and a high-reactive F1 hybrid was used. Highly significant linkage was established only with regard to the H-2 marker but no strong linkage was found with nine other markers segregating in this cross. Recent results confirmed the influence of H-2 linked factors on NK activity (8). Heterozygosity within or near the H-2D region was sufficient to ensure in vivo resistance as well as high NK reactivity in vitro against the EL-4 lymphoma.

References

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GENERATION IN VIVO OF MOUSE NATURAL CYTOTOXIC CELLS

Otto Haller

Publisher Summary

This chapter provides an overview of the generation of mouse natural cytotoxic cells in vivo. As the mouse is a convenient species for in vivo manipulation, the generation and maturation of mouse natural killer cells (NK) was explored using various experimental approaches. The notable features of the NK system reported by most investigators are as follows: NK activity (1) shows distinct organ distribution; (2) is largely independent of a functional thymus; and (3) changes with the age of the individual mouse in a manner quite different from B-and T-lymphocyte function. Furthermore, NK cell activity is under strict genetic control with the classification of inbred mouse strains as NK high or low reactive. The mechanisms through which NK activity is governed are largely unknown. Neither spleen nor thymus seems to be involved in the differentiation/maturation process leading to NK cells. NK cells might gain specific cytotoxicity as a result of exposure and sensitization to ubiquitous antigens present in the host environment. Alternatively, the degree of NK cell activity might be genetically preprogrammed at the NK precursor cell level. It would, thus, seem that the in vivo generation of NK cells is an inborn and autonomous function controlled by stem cells present in marrow or fetal liver. NK cell generation would seem not to depend on the genotype or other influences of the host