Publisher Summary Studies of cytotoxicity by human lymphocytes revealed not only that both allogeneic and syngeneic tumor cells were lysed in a non-MHC-restricted fashion, but also that lymphocytes from normal donors were often cytotoxic. Lymphocytes from any healthy donor, as well as peripheral blood and spleen lymphocytes from several experimental animals, in the absence of known or deliberate sensitization, were found to be spontaneously cytotoxic in vitro for some normal fresh cells, most cultured cell lines, immature hematopoietic cells, and tumor cells. This type of nonadaptive, non-MHC-restricted cellmediated cytotoxicity was defined as “natural” cytotoxicity, and the effector cells mediating natural cytotoxicity were functionally defined as natural killer (NK) cells. The existence of NK cells has prompted a reinterpretation of both the studies of specific cytotoxicity against spontaneous human tumors and the theory of immune surveillance, at least in its most restrictive interpretation. Unlike cytotoxic T cells, NK cells cannot be demonstrated to have clonally distributed specificity, restriction for MHC products at the target cell surface, or immunological memory. NK cells cannot yet be formally assigned to a single lineage based on the definitive identification of a stem cell, a distinct anatomical location of maturation, or unique genotypic rearrangements.

Biology of natural killer cells.

Publisher Summary This chapter focuses on the important discovery that virus-specific cytotoxic T cells are dually specific for virus and for a self cell surface antigen encoded by the major histocompatibility complex (MHC). The initial work was carried out on the lymphocytic choriomeningitis virus system but it soon became evident that the same phenomenon applied to many other viruses. In addition, the same principle has been found to hold for other antigenic systems, such as trinitrophenyl coupled to cells, minor histocompatibility antigens, and the H-Y model. Graft rejection and the need for genetically homogeneous inbred mouse strains for cancer research led to the development of transplantation immunology and immunogenetics. The result is that the gene complex coding for major transplantation antigens is one of the better understood mammalian genetic regions. Cytotoxic T-cell specificity is comparable to serological specificity. Because quantification of specificity or cross-reactivity is difficult, and because of the technical limitations of these cytotoxic T-cell assays, results are interpreted with great reservation. MHC restriction reflects the fact that the effector function of T cells is determined by the kind of Self-H recognized together with the foreign antigen on cell surfaces: K and D are receptors for lytic signals, I determinants are receptors for cell differentiation signals that are delivered antigen-specifically by T cells.

MHC-restricted cytotoxic T cells: studies on the biological role of polymorphic major transplantation antigens determining T-cell restriction-specificity, function, and responsiveness.

In the spleens of young, adult mice there exist naturally occurring killer lymphocytes with specificity for mouse Moloney leukemia cells. The lytic activity was directed against syngeneic or allogeneic Moloney leukemia cells to a similar extent, but was primarily expressed when tested against in vitro grown leukemia cells. Two leukemias of non-Moloney origin were resistant and so was the mastocytoma line P815. Although killer activity varied between different strains of mice, the specificity of lysis was the same as indicated by competition experiments using unlabeled Moloney or other tumor cells as inhibitors in the cytotoxic assays. Capacity to compete and sensitivy to lysis by the killer cells were found to be highly positively correlated. Analysis of the kinetics of the cytotoxic assay revealed a rapid induction of lysis within one to four hours, arguing against any conventional in vitro induction of immune response. No evidence was found of soluble factors playing any role in the cytolytic assay.

"Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype.

Lymphoid cells from many normal mice of a variety of inbred strains were found to have reactivity, in a 51Cr release cytotoxicity assay, against several syngeneic and allogeneic tumors. Very high reactivity was seen with effector cells from athymic nude mice, which was consistent with other evidence that the reactivity was not T-cell dependent. Target cells susceptible to lysis included tumors induced by oncogenic type-C viruses but also tumors induced by other means and expressing endogenous type-C viruses. The levels of natural reactivity were influenced by age, with highest cytotoxicity produced by cells from 5- to 8-week-old mice. Lymph-node cells, spleen cells, peritoneal exudate cells and peripheral blood lymphocytes all had cytotoxic reactivity. The specificity of the reactions was analyzed in detail by ana inhibition assay. Evidence was obtained for natural reactivty against several different antigens, each apparently associated with expression of murine endogenous type-C viruses.

Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. I. Distribution of reactivity and specificity

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 Cell-Mediated Immunity

Destruction of Human Lymphoid Tissue-Culture Cell Lines by Human Peripheral Lymphocytes in 51Cr-Release Cellular Cytotoxicity Assays

Role of Suppressor Cells in Depression of In Vitro Lymphoproliferative Responses of Lung Cancer and Breast Cancer Patients

Inhibition of Leukocyte Migration by Tumor-Associated Antigens in Soluble Extracts of Human Breast Carcinoma

Adherent-phagocytic cells were obtained from the peripheral blood and the unstimulated peritoneal cavity of normal C3H/HeN mice and tested for their cytotoxicity in an in vitro 3 H-TdR release assay and in a growth inhibition assay. These cells had appreciable cytolytic and cytostatic activity, which varied with the attacker: target (A:T) cell ratio, against TU5, an SV40 virus-transformed BALB/c tumor cell line. Cytolytic activity was first detected after 48 hr of incubation and was considerably higher at 72 hr. In contrast, this line was resistant to cytotoxicity by nonadherent lymphoid cells at the A:T ratios employed, and adherent cells from the peritoneal cavity were not cytotoxic against YAC-1 target cells in a 4-hr 51 Cr release assay. The natural cytotoxicity of adherent cells from the peritoneal cavity was largely inhibited by treatment with silica. These results indicated that the natural reactivity observed here was mediated by macrophages and not by natural killer cells. The cytotoxic reactivity of the macrophages from normal mice could not be attributed to activation by a variety of possible in vivo or in vitro stimuli. Cells from germ-free nude mice had cytolytic activity, as did cells cultured in medium containing endotoxin-free fetal bovine serum or normal mouse serum. Activation by interferon produced in vitro was also unlikely since marcophages cultured in the presence of anti-interferon were also cytotoxic. As previously described for activated macrophages, peritoneal macrophages from normal C3H/HeJ mice were significantly less cytotoxic than macrophages from syngeneic C3H/HeN mice. The monocytes and macrophages seemed to have somewhat different properties since the addition of lymphokine-containing medium to the adherent peripheral monocytes increased their cytotoxicity, whereas no enhanced activity was observed by adding this supernatant to resident peritoneal macrophages. The normal macrophages and monocytes reacted against both syngeneic (1023) and allogeneic (TU5) tumor cells as well as mouse fibroblast cells transformed by SV40 virus (SV40-3T3). Similar activity was also observed against a human cell line derived from a patient with breast cancer (G11) and against human fibroblast cells transformed by SV40 (SV40-2931 and SV40-2303), but not against nontransformed fibroblasts from the same donors. Thus, normal murine peripheral monocytes and peritoneal macrophages possess natural cytotoxic activity that is selective for tumor and virus-transformed cells.

Natural cytotoxicity of mouse monocytes and macrophages.

Lymphocyte proliferation (LP) assays were performed in microculture using the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A); the T + B cell mitogens, pokeweed mitogen (PWM) and staphylococcal phage lysate (SPL); and a pool of allogeneic stimulating leukocytes in one-way mixed leukocyte cultures (MLC) in lung and breast cancer patients and normal individuals. The resultant data were expressed in three different ways: (1) as mean counts per minute (CPM) of tritiated thymidine incorporation; (2) as a stimulation index (SI) and (3) as a relative proliferation index (RPI). The RPI is defined as the ratio of net CPM (nCPM) in experimental cultures with stimulant (E) minus medium control cultures (C) of a test individual to the mean nCPM of three or more normal individuals examined in the same assay on the same day. These expressions were then compared for their ability to discriminate between LP responses in cancer patients and normal individuals. The RPI value and selected cut-off values gave the most sensitive measure for the determination of depressed proliferative responses. These analyses demonstrated that lung carcinoma patients were depressed to PHA (50%), MLC (47%), PWM (43%) and Con A (40%). To a lesser degree, breast carcinoma patients were also depressed to MLC (36%), PHA (31%), PWM (27%) and Con A (19%). Our data indicate that the use of the RPI in the analysis of LP response represents an improved method for detecting impaired response of lymphocytes to general mitogens and alloantigens which can consistently reveal immunosuppression in many cancer patients and may be useful for serial monitoring of individual patients.

The relative proliferation index as a more sensitive parameter for evaluating lymphoproliferative responses of cancer patients to mitogens and alloantigens.

J. L. McCoy

Papers

Destruction of Human Lymphoid Tissue-Culture Cell Lines by Human Peripheral Lymphocytes in 51Cr-Release Cellular Cytotoxicity Assays

Role of Suppressor Cells in Depression of In Vitro Lymphoproliferative Responses of Lung Cancer and Breast Cancer Patients

Inhibition of Leukocyte Migration by Tumor-Associated Antigens in Soluble Extracts of Human Breast Carcinoma

Natural cytotoxicity of mouse monocytes and macrophages.

The relative proliferation index as a more sensitive parameter for evaluating lymphoproliferative responses of cancer patients to mitogens and alloantigens.