Relationship between cell-mediated and humoral immune attack on tumor cells. I. Drug and hormone effects on susceptibility to killing and macromolecular synthesis

The effect of metabolic inhibitors and hormones on the susceptibility of P815 murine mastocytoma cells to antibody-C and cell-mediated killing, and on the ability of the cells to synthesize DNA, RNA, protein, carbohydrate, and lipid was tested. Pretreatment of the cells with adriamycin, actinomycin D, and puromycin increased the sensitivity of the cells to killing by rabbit anti-P815 antibody plus guinea pig C, but not by allogeneic P815-sensitized spleen cells. Conversely, mitomycin C treatment enhanced the cells' sensitivity to cell-mediated, but not antibody-C, killing. Insulin and hydrocortisone, but not epinephrine, were effective in decreasing the susceptibility of the cells to killing by both antibody-C and cell-mediated attack systems. The kinetics of the drug-induced increase and the hormone-induced decrease in susceptibility of the cells to antibody-C killing correlated with a decrease and increase, respectively, in the ability of the cells to incorporate fatty acid into complex cellular lipid. No such correlation was found between cellular lipid synthesis and tumor cell susceptibility to cell-mediated killing. The ability of the cells to resist either form of immune attack was not dependent on the ability of the cells to synthesize DNA, RNA, protein, or complex carbohydrate. These results suggest that the susceptibility of these tumor cells to antibody-C vs cell-mediated killing may be linked to different metabolic properties of the cells, and may reflect differences in the mechanisms of humoral vs cellular immune attack.     

Identification of lipids associated with the ability of tumor cells to resist humoral immune attack.

Certain metabolic inhibitors or chemotherapeutic agents that increase the susceptibility of line-1 or line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of the cells to synthesize lipids. A direct correlation was found between the drug-induced increase in sensitivity to antibody-C mediated killing and the inhibition of the ability of the cells to incorporate acetate, glycerol, and fatty acids into complex cellular lipids. Drug-treated cells recultured in drug-free medium regained their resistance to antibody-C mediated killing; these cells recovered their ability for complex lipid synthesis at this time. Thin layer chromatography of CHCl3:CH3OH lipid extracts from these cells indicated that the drug-induced increase in susceptibility to humoral immune attack correlated with the inhibition of acetate, glycerol, and fatty acid incorporation into cardiolipin and triglyceride in line-10 cells and the inhibition of incorporation of these compounds into cardiolipin alone in line-1 cells. No direct correlation was found between the sensitivity of the cells to humoral immune attack and the ability of the cells to incorporate precursors of lipid synthesis into other lipid moieties (sphyngomyelin, phosphatidyl serine, phosphatidyl choline, phosphatidyl glycerol, or cholesterol esters). The synthesis of cardiolipin and triglycerides, therefore, appears to be associated with the mechanism whereby these tumor cells resist antibody-C mediated killing.     

Correlation between the ability of tumor cells to resist humoral immune attack and their ability to synthesize lipid.

Agents that increase (certain metabolic inhibitors, chemotherapeutic agents, and x-irradiation), decrease (hormones), or have no effect (hyperthermia) on the susceptibility of line-1 and line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of these tumor cells to synthesize macromolecules. A correlation was found between the drug-induced increase in sensitivity of these cells to antibody-C mediated killing and the loss of their ability to incorporate fatty acids into complex cellular lipids. Similarly, the hormone-induced increase in resistance of the cells to killing was accompanied by an enhancement in complex lipid synthesis by these cells was also observed after the cells were exposed to physical means of insult (x-irradiation or hyperthermia). No correlation was found between the sensitivity of the cells to antibody-C mediated killing and their ability to synthesize DNA, RNA, protein, or complex carbohydrate, or their capacity for de novo lipid synthesis as measured by incorporation of acetate and glycerol into cellular macromolecules. The assembly of free fatty acids into complex lipid moieties is therefore proposed to be of fundamental importance for the ability of the tumor cells to resist humoral immune killing.     

Physico-chemical properties of tumor cells that influence their susceptibility to humoral immune killing

Line-10 guinea pig hepatoma cells are susceptible to killing by antibody plus human complement, but resistant to killing by antibody plus guinea pig complement. Tumor cells treated with agents that reversibly increase (adriamycin), decrease (insulin or hydrocortisone), or have no effect (5-fluorouracil) on the susceptibility of the cells to antibody-complement killing were tested for their lipid and fatty acid composition. Hormone-treated (resistant) cells showed an increased total lipid content, an increased cholesterol: phospholipid ratio, and a depressed level of unsaturated fatty acids in the cellular neutral and phospholipids compared to control untreated cells. Adriamycin-treated (sensitive) cells showed exactly the opposite effects. The lipid composition of both hormone- and adriamycin-treated cells returned to control levels when the cells reverted to control levels of susceptibility to antibody-complement killing. 5-fluorouracil-treated cells were indistinguishable from untreated controls in their lipid and fatty acid composition. The chemical composition of the cell, and its effects on the physical properties of the cellular membranes, therefore appears to be fundamental for the ability of these tumor cells to resist humoral immune attack.     

Specific 125I-iodination of cell surface lipids: plasma membrane alterations induced during humoral immune attack.

A method whereby lactoperoxidase-catalyzed 125I-iodination of plasma membrane lipids can be achieved is described. The reaction results in a uniform and stable labeling of neutral lipids, phospholipids, lysophosphatides, free fatty acids, and triacylglycerides. By the use of this method, the action of antibody plus complement (C) on the specific release of lipid from the plasma membrane of line-10 tumor cells was studied. Within 15 min after the addition of C to antibody-sensitized cells, the enhanced release of specific lipid classes from the cell surface was observed; these lipids included sphingomyelin, phosphatidylserine, and phosphatidylcholine. The release of phosphatidylethanolamine and, in some instances, triglycerides, was reduced after antibody-C attack. Neither the specificity of the antibody used to sensitize the cells nor the ability of the antibody plus C to be cytotoxic to the cells appeared to affect the identity or amounts of lipids released from the cells.     

Stimulation of the synthesis and release of lipids in tumor cells under attack by antibody and C

Antibody-sensitized line-1 or line-10 tumor cells treated with GPC (TAC) incorporated fatty acids into complex cellular lipids and released increased amounts of fatty acids within 5 to 10 min after the addition of GPC as compared to control cells. This effect was dependent on the concentration of GPC used; however, under conditions where the cells were not killed, the enhanced synthesis and release of lipids were not dependent on the antibody concentration used to sensitize the cells. Treatment of the cells with antibody alone, GPC alone, or antibody plus heat-inactivated GPC did not result in enhanced synthesis or release of lipids. No enhancement in DNA, RNA, or protein synthesis in TAC was noted. Line-1 cells, which can be killed by GPC when sensitized with excess anti-Forssman IgM antibody, demonstrated enhanced lipid synthesis within 1 to 3 min after the addition of GPC to the antibody-sensitized cells, before measurable killing of the cells had occurred. This effect persisted in the surviving cells when tested 5 and 10 min after the formation of TAC. Addition of GPC deficient in C4 to antibody-sensitized cells did not result in enhanced lipid synthesis or release. These data suggest that the synthesis of macromolecules of which lipids are a major component is of central importance for the ability of the cells to resist antibody-GPC mediated attack.     

Exposure to paclitaxel or vinblastine down-regulates CD11a and CD54 expression by P815 mastocytoma cells and renders the tumor cells resistant to killing by nonspecific cytotoxic T lymphocytes induced with anti-CD3 antibody

Paclitaxel and vinblastine are drugs with anti-microtubule activity that are commonly used in the treatment of numerous types of cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel or vinblastine on the subsequent susceptibility of surviving P815 murine mastocytoma cells to cytolysis by major histocompatibility complex (MHC)-unrestricted mouse cytotoxic T lymphocytes that had been induced with anti-CD3 antibody. P815 cells that had survived culture for 24 h in the presence of paclitaxel (5 or 50 micro g/ml) or vinblastine (1.5 or 15 micro g/ml) were rendered resistant to anti-CD3-activated killer-T (AK-T) cell-mediated cytolysis in a standard (51)Cr-release assay. Resistance to killing was associated with a reduced ability of AK-T cells to form conjugates with drug-treated P815 target cells, suggesting a possible effect on adhesion molecules. Flow cytometric analysis of paclitaxel- or vinblastine-treated P815 cells revealed reduced cell-surface expression of the adhesion molecules LFA-1 (CD11a /CD18) and ICAM-1 (CD54). Similar results were obtained following paclitaxel or vinblastine treatment of Yac-1 lymphoma cells. RT-PCR analysis revealed reduced levels of mRNAs coding for CD11a and CD54 in paclitaxel- or vinblastine-pretreated P815 cells. Collectively, these data lead us to conclude that paclitaxel and vinblastine render P815 mastocytoma cells resistant to T cell-mediated cytotoxicity by interfering with CD11a and CD54 expression by the tumor cells. A similar effect by these drugs on tumor cells and/or leukocytes in cancer patients might compromise tumor-specific cell-mediated immune responses.     

Distinct restriction of complement- and cell-mediated lysis

Complement- and cell-mediated killing utilize related effector proteins (C8/C9 and perforin, respectively), suggesting that proteins which protect cells against complement- and cell-mediated attack may also be similar. In homologous complement-mediated killing two protective proteins, which are anchored to the cell membrane by phosphatidylinositol glycan (PIG) tails, are known. To study whether similar PIG-tailed proteins protect against lymphocyte-mediated killing, nucleated cell lines with a mutation in the biosynthesis of the PIG anchor were used. It was found that PIG-tailed membrane proteins restrict homologous complement-mediated lysis but not three different types of cell-mediated killing or lysis by purified perforin. Furthermore, E from patients with an acquired defect in PIG tail biosynthesis did not differ from normal E in sensitivity to antibody-dependent cell-mediated cytotoxicity, in spite of their increased sensitivity to human C8 and C9.     

Liposomes as model membrane systems for immune attack. I. Transfer of antigenic determinants to lymphocyte membranes after interactions with hapten-bearing liposomes.

The interaction of human peripheral blood lymphocytes with liposomes containing DNP-aminocaproyl-phosphatidylethanolamine together with either egg yolk or dipalmitoylphosphatidylcholine has been investigated. When lymphocytes were incubated with liposomes at 37 degrees C, the aqueous compartment (86Rb+) and the lipid portion (3H-lipid) of the liposomes became cell associated to an equivalent extent. At 0 degrees C, however, the incorporation of 3H-lipid exceeded that of 86Rb+. Lymphocyte-liposome interactions were accompanied by the transfer of DNP to the surface of the lymphoid cell as measured by susceptibility to complement in the presence of anti-DNP antibody. Hapten transfer was not limited to liposome interactions with lymphocytes, but occurred also with other cells (e.g., Chang cells). Hapten transfer could also be demonstrated by susceptibility to K cell-mediated lysis. These findings suggest that liposomes may prove to be a useful vehicle for the transfer of new antigenic determinants onto cell surfaces. The implications of these findings are discussed in the context of using liposomes as targets for cell-mediated cytotoxic attack.     

Macrophages help NK cells to attack tumor cells by stimulatory NKG2D ligand but protect themselves from NK killing by inhibitory ligand Qa-1

Natural killer (NK) cells and their crosstalk with other immune cells are important for innate immunity against tumor. To explore the role of the interaction between NK cells and macrophages in the regulation of anti-tumor activities of NK cells, we here demonstrate that poly I:C-treated macrophages increased NK cell-mediated cytotoxicity against target tumor cells in NKG2D-dependent manner. In addition, IL-15, IL-18, and IFN-β secreted by poly I:C-treated macrophages are also involved in NKG2D expression and NK cell activation. Interestingly, the increase in expression of NKG2D ligands on macrophages induced a highly NK cell-mediated cytotoxicity against tumor cells, but not against macrophages themselves. Notably, a high expression level of Qa-1, a NKG2A ligand, on macrophages may contribute to such protection of macrophages from NK cell-mediated killing. Furthermore, Qa-1 or NKG2A knockdown and Qa-1 antibody blockade caused the macrophages to be sensitive to NK cytolysis. These results suggested that macrophages may activate NK cells to attack tumor by NKG2D recognition whereas macrophages protect themselves from NK lysis via preferential expression of Qa-1.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5°C, multiplying at 33°C) and cold-sensitive (arrested at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in ‘gently lysed cells’ (obtained by treatment with 0.01% Brij-58) or ‘highly lysed cells’ (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [³H]dTTP efficiently, while highly lysed cells exhibited a low level of [³H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extracts reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.     

Hemorrhage induces enhanced Kupffer cell cytotoxicity while decreasing peritoneal or splenic macrophage capacity. Involvement of cell-associated tumor necrosis factor and reactive nitrogen.

Studies indicate that simple hemorrhage produces a profound depression of cell-mediated immunity, thereby contributing to an enhanced susceptibility to septic challenge in the host. However, it remains unknown whether or not the macrophages' cytotoxic capacity is altered after hemorrhage. To study this, C3H/HeN mice were bled to and maintained at a blood pressure of 35 mm Hg for 60 min, and adequately resuscitated. Mice were then killed at 2 or 24 h after hemorrhage to obtain peritoneal macrophage, splenic macrophage, and Kupffer cells. Cytotoxicity was assessed by determining the capacity of these macrophages to lyse [3H]TdR labeled WEHI-164 clone 13 or P815 tumor target cells (WEHI-164, sensitive to both soluble and cell-associated TNF vs P815 cells, insensitive to soluble TNF). Peritoneal and splenic macrophages from hemorrhaged animals exhibited a significantly reduced cytotoxic capacity, whereas Kupffer cells' ability to kill the target cells was enhanced. Similarly, the Kupffer cells' capacity to release TNF and IL-1, as well as express cell-associated forms of this cytokine are significantly enhanced on macrophages isolated 2 h after hemorrhage, whereas peritoneal macrophages are not. Furthermore, antibodies directed at mouse TNF but not against murine IL-1 alpha or murine IL-6 were able to oblate the enhanced target cell lysis of unfixed, as well as paraformaldehyde fixed (metabolically inactive) Kupffer cells. Studies using inhibitors (GN-monomethyl-arginine, superoxide dismutase, catalase, and ibuprofen) of other TNF-inducible mechanisms of target cell killing indicated that only the inhibition of the release of reactive nitrogen consistently depressed the cytotoxic capacity of Kupffer cells from hemorrhaged mice. Thus, the increased Kupffer cell cytotoxicity from hemorrhaged mice is most likely mediated through the expression of cell-associated TNF and the release of reactive nitrogen.     

Failure of specific adoptive immunotherapy owing to survival and outgrowth of variant cells

Summary Adoptive immunotherapy, the transfer of spleen cells from immunized mice to mice with a small tumor, was usually curative for mice with the P815 mastocytoma provided that steps were taken to prevent the generation of tumor-induced suppressor cells in the recipient animal. However, failure of adoptive immunotherapy of the P815 tumor, resulting in regrowth of either the primary intradermal or a metastatic tumor, was observed in 10 out of 112 animals receiving graded doses of 7.5×10⁷ to 3.0×10⁸ immune spleen cells. Examination of the ten tumors in mice that failed to respond to therapy revealed that seven of them were significantly less susceptible than the original P815 tumor to rejection in vivo by transferred anti-P815-specific effector cells. In addition, nine of the ten therapy-failure tumors were also less susceptible than the original P815 tumor to lysis in vitro by P815-specific, but not DBA/2-specific, cytotoxic T lymphocytes. Sensitivity to lysis by tumor-specific cytotoxic T cells was not, however, strongly correlated with sensitivity to rejection in vivo by P815-specific effector spleen cells. Neither in vivo sensitivity to rejection, nor sensitivity to cytotoxic T cells, was correlated with alterations in class I major histocompatibility complex antigen expression. These results suggest that the survival and outgrowth of variant tumor cells was frequently the cause of failure of specific adoptive immunotherapy of the P815 tumor, and that selection for cells with a reduced sensitivity to killing by cytotoxic T cells was only one mechanism that might lead to an immunotherapeutic failure.This work was supported by a grant from RJR Nabisco Inc., a grant from the J. M. Foundation, and by USPHS grant CA-40597 awarded by the National Cancer Institute     

Conditions controlling tumor cytotoxicity of rat liver macrophages mediated by liposomal muramyl dipeptide

Activation of rat liver macrophages with free and liposome-encapsulated muramyl dipeptide (MDP) to a tumorcytotoxic state was characterized by employing various experimental conditions. Macrophage-mediated tumor cytotoxicity was determined using two standard assay systems: a [methyl-3H]thymidine release assay to measure the extent of tumor cell lysis and a [methyl-3H]thymidine incorporation assay to measure the combined effects of tumor cell lysis and stasis. The extent of cell lysis was not affected by the ratio of macrophages to tumor cells within the ratio range of 30:1 to 5:1, provided that the macrophages form a confluent monolayer. Tumor cell lysis, however, was significantly influenced by macrophage density; a low macrophage density for example resulted in a low percentage of tumor cell lysis. Tumor target cells used in this study, i.e., C26 adenocarcinoma, B16 melanoma and P815 mastocytoma, differed in their susceptibility towards macrophage-mediated cell lysis, whereas no differences were observed with respect to tumor cell stasis. Non-tumorigenic cell lines such as human fibroblastic cells and LLC monkey kidney cells were not lysed by activated macrophages, although proliferation of these cells was markedly inhibited. Additionally, the effects of liposomal lipid composition on macrophage activation were studied. With a basic composition of phospholipid/cholesterol/dicetylphosphate, we used either egg-yolk, dipalmitoyl-, distearoyl- or dihexadecylphosphatidylcholine as the bulk phospholipid constituent. Although these liposomes display a widely different susceptibility to lysosomal phospholipase activities, we could not detect any significant difference in either the extent or the duration of the tumoricidal activity induced by MDP encapsulated in these different types of liposomes.     

Intracamerally induced concomitant immunity: mice harboring progressively growing intraocular tumors are immune to spontaneous metastases and secondary tumor challenge

Intracameral inoculation of allogeneic P815 mastocytoma cells (DBA/2) into BALB/c mice resulted in progressively growing intraocular tumors. Intraocular tumor cells disseminated rapidly to the spleen and cervical lymph nodes, yet extraocular nests of tumor cells never developed into fulminant tumors. Further experiments showed that tumor cells were continuously seeded from the primary intraocular tumor and were rapidly cleared from extraocular sites. Hosts harboring intraocular P815 mastocytomas rejected tumorigenic doses of P815 cells inoculated subcutaneously or even into the contralateral anterior chamber. This systemic tumor immunity was found to be radiosensitive and T cell dependent. Spleen cells from animals with progressively growing intraocular tumors protected recipient mice challenged with intracamerally inoculated tumor cells and thus suggests that a cell-mediated mechanism is the underlying basis for this form of tumor immunity. The data indicate that mice harboring progressively growing intraocular tumors develop a potent state of "concomitant immunity," that prevents the development of metastases, yet is ineffective in controlling the primary tumor.     

IFN-gamma differentially modulates the susceptibility of L1210 and P815 tumor targets for macrophage-mediated cytotoxicity. Role of macrophage-target interaction coupled to nitric oxide generation, but independent of tumor necrosis factor production

IFN-gamma primes murine macrophages to render them responsive for triggering by subactivating concentrations of bacterial LPS to mediate nonspecific tumor cytotoxicity. However, IFN-gamma also has direct anti-proliferative effects on transformed cells that serve as sensitive tumor targets for cytotoxic macrophages. We investigated the effects of preexposure of L1210 mouse leukemia and P815 mouse mastocytoma targets to rIFN-gamma on changes in their susceptibility to cytotoxicity by LPS-activated mouse peritoneal macrophages (PM). Co-incubation of inflammatory PM and either L1210 or P815 targets with IFN-gamma and LPS produced a classical synergistic cytotoxicity for both targets over that of IFN-gamma or LPS alone. Similar synergistic augmentation of cytotoxicity occurred when effector PM were preprimed for 24 h with IFN-gamma before testing for cytotoxicity of untreated targets. However, pretreatment of L1210 and P815 targets for 24 h with IFN-gamma (50 U) before assay produced divergent results in that L1210 was more susceptible, whereas P815 was less susceptible to cytotoxicity by LPS-activated macrophages. Similar results were obtained when both macrophages and targets were pretreated separately with IFN-gamma for 24 h before their combined assay for tumor cytotoxicity. Pretreatment of L1210 targets for 1, 4, or 24 h with IFN-gamma produced similar effects on their increased susceptibility to macrophage cytotoxicity. In contrast, P815 pretreated for 1 and 4 h with IFN-gamma showed an early increased susceptibility to macrophage cytotoxicity followed by a decrease after 24 h pretreatment. The pretreatment of L1210 or P815 targets with IFN-gamma before their exposure to LPS-activated macrophages had no effect on the production of TNF. However, there was a corresponding increase in nitric oxide generation by LPS-activated macrophages after their exposure to IFN-gamma pretreated L1210 targets and a decrease in the presence of IFN-gamma-pretreated P815 targets that correlated with their changes in susceptibility to macrophage killing. Nitric oxide generation by macrophages alone in response to LPS was found to be greater than when effector macrophages were exposed to the tumor targets and this was either increased by L1210 or decreased by P815 that had been pretreated with IFN-gamma. Our results indicate that IFN-gamma may act directly and differentially on tumor targets to alter their susceptibility for macrophage cytotoxicity, which was coupled to changes in the generation of cytotoxic nitric oxide, rather than TNF production by the macrophage.(ABSTRACT TRUNCATED AT 400 WORDS)     

NK cells and cancer

In this review, we overview the main features and functions of NK cells, focusing on their role in cell-mediated immune response to tumor cells. In parallel, we discuss the information available in the field of NK cell receptors and offer a wide general overview of functional aspects of cell targeting and killing, focusing on the recent acknowledgments on the efficacy of NK cells after cytokine and mAb administration in cancer therapy. Since efficacy of NK cell-based immunotherapy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands, as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell-based immunotherapy.     

Pretreatment of P815 mastocytoma cells with inhibitors of protein synthesis reduces their susceptibility to lysis by cytotoxic thymus-derived lymphocytes

Protein synthesis inhibitors like cycloheximide, emetine, and puromycin diminish the ability of P815, a mastocytoma of DBA/2 mice, to react with anti-H-2^d cytotoxic thymus-derived lymphocytes (CTLs). Compared to untreated P815, tumor cells incubated with the protein synthesis inhibitors exhibited a reduced sensitivity to lysis and a reduced ability to inhibit lysis of untreated P815 cells. Consistent with this reduced reactivity of cycloheximide-treated P815 cells with CTLs was the inability of anti-H-2^d CTLs to form T cell-target cell conjugates with treated P815 cells. As evaluated by the binding of an anti-H-2^d serum, treated P815 cells expressed the same amount of H-2 membrane antigen as untreated cells. However, treated cells were still lysed by CTLs in the presence of the agglutinator, concanavalin A (Con A).     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.