Immune and natural antibodies to syngeneic murine plasma cell tumors.

Cytotoxic antibody to a plasma cell tumor antigen was produced in syngeneic BALB mice by immunization with viable or inactivated plasma cell tumors. Antibody with the same specificity was found in the sera of normal BALB and other strains of mice. This natural antibody reacted with an antigen with characteristics indistinguishable from the previously described alloantigen, PC.1, and with viral envelope antigen, chiVEA. The incidence of cytotoxic reactivity and the antibody titers reached a peak in normal BALB mice at 3-4 months of age, and were lower in 9-12-month old mice. The sera of germfree mice had lower reactivity; but when the mice were transferred to conventional conditions, their sera soon became as active as those of conventional mice. A virus common to all plasma cell tumors, which is present in latent form in some normal tissues of BALB and other PC.1 positive strains, is suggested as the cause for the PC.1 antigen and for the appearance of natural antibody to it. The considerable evidence for the close association of a virus with plasma cell tumors is presented.

Production of plasminogen activator by human natural killer cells. Large granular lymphocytes.

In this report we have used highly purified populations of natural killer (NK) cells: large granular lymphocytes (LGL). This study demonstrates that freshly isolated and interleukin 2-cultured LGL produce the specific neutral serine protease, plasminogen activator (PA). We have found that the enzyme is expressed in both an extracellular form as well as in a cell-associated form. Upon subcellular distribution the latter form of the enzyme is associated with a cell-surface membrane-enriched fraction. LGL PA exists in multiple molecular weight forms ranging from 100,000 to 26,000. Interferon (IFN), the major positive regulator of NK cytolytic activity, caused a substantial enhancement of cell-associated, but not extracellular, PA. In contrast, LGL isolated from patients with Chediak-Higashi syndrome, who are known to be defective in NK activity, displayed low PA activity, altered morphology, and low NK killing relative to LGL isolated from normal donors. The possible role of LGL PA in the lysis of tumor cells by NK cells, either directly or indirectly, is discussed.

Characteristics of human large granular lymphocytes and relationship to natural killer and K cells.

Recent evidence, has demonstrated an association between a subpopulation of peripheral blood mononuclear cells, morphologically identified as large granular lymphocytes (LGL), and natural killer (NK) activity. We have now evaluated more directly the role of LGL in both NK activity and antibody- dependent cellular cytotoxicity (ADCC), by using highly enriched populations of LGL, obtained by centrifugation of peripheral blood mononuclear cells on Percoll discontinuous density gradients. Both spontaneous and interferon- augmented NK and ADCC activities were exclusively associated with the LGL- enriched, low density fractions. The majority of LGL formed conjugates with NK-susceptible and antibody-coated target cells. Approximately 20 percent of small conventional lymphocytes also formed conjugates with the target cells for NK, but this was not associated with cytotoxic activity. Virtually all LGL were found to have receptors for the Fc portion of IgG (FcgammaR). The frequency of LGL among blood leukocytes was 2-6 percent. LGL could be enriched to an average purity of 95 percent by combining discontinuous density gradient centrifugation with subsequent adsorptions of the low density fractions on monolayers of immobilized immune complexes. About 50 percent of LGL were found to be FcgammaR-bearing T cells (T(G)), forming low affinity rosettes with sheep erythrocytes at 4 degrees C. Only 10-20 percent of LGL formed high affinity rosettes with sheep erythrocytes at 29 degrees C. LGL could be enriched to a purity of more than 90 percent by depleting high affinity rosette-forming cells from low density Percoll fractions. LGL were only a subpopulation of T(G) cells, because some lymphocytes with conventional morphology also adhered to the immobilized immune complex monolayers and formed high affinity rosettes with sheep erythrocytes. Separation of these cells from LGL by discontinuous density gradient centrifugation indicated that they are not cytotoxic, suggesting a morphological and functional subdivision of T(G) cells. The verification in this study that virtually all human NK and K cells have a characteristic morphology adds a useful parameter to the monitoring of human lymphocytes, and the ability to purify these cells by simple physical procedures should be invaluable in their further characterization.

In vitro activation of cellular immune response to Gross virus-induced lymphoma.

Spleen cells from W/Fu rats 40 days or more after immunization with a syngeneic Gross virus-induced leukemia were unreactive in direct cytotoxic assays. Incubation of these immune cells at 37 degrees C for 12 hr or longer, in the absence of antigen, resulted in the appearance of specific cytotoxic reactivity. Other lymphoid cells from the immune rats also were activated upon in vitro incubation, but to a lesser extent. Experiments were performed to define the necessary conditions and the mechanism for the in vitro incubation. Activation was temperature dependent, occurring at 37 degrees C but not at 4 degrees C. Immune serum suppressed the activation, but normal rat serum also had some inhibitory activity. Passage of immune cells through a nylon column, before preincubation, prevented activation. In contrast, exposure to nylon after preincubation did not remove cytotoxic reactivity. These findings demonstrate the reversal of a central inhibition of immune cell activity. The explanations offered for this phenomenon included change in surface characteristics of the immune cells during in vitro incubation, and the possible need for an adherent helper cell.

Augmentation of mouse natural killer activity and induction of interferon by tumor cells in vivo.

Conventional and nude mice inoculated with syngeneic or allogenic tumor cells developed a rapid rise in serum interferon (IF) levels, peaking within 24 h. Within the same period, natural killer (NK) activity was readily boosted in the spleen. Both activities usually declined at 3 d. Cells that lacked the ability to augment NK activity also failed to induce detectable levels of IF. The boosting of IF and NK functions did not appear to be a result of contamination of the tumor lines by viruses because inoculation of several type C viruses into normal mice had no effect, and other viruses, like lymphocytic choriomeningitis virus and influenza, elevated IF and NK levels with a significantly later kinetics, peaking 3-4 d. The IF induced by tumor cells was heat and acid labile, species specific, and appeared to be in the type II class, although it was susceptible to antisera against Newcastle disease virus-induced IF. These data suggest that an early, nonthymus-dependent consequence of tumor-cell recognition is the production of IF, which, in turn, activates NK cells to lyse the tumor cells.

Lymphokine-activated killer cells in rats. III. A simple method for the purification of large granular lymphocytes and their rapid expansion and conversion into lymphokine-activated killer cells.

A simple method for the purification and rapid expansion of large granular lymphocytes into cells with efficient broad antitumor cytotoxicity after stimulation by human rIL-2 is described. Nylon-wool nonadherent splenic mononuclear leukocytes from Fischer 344 rats were cultured in medium containing 1,000 U/ml rIL-2. The initial response of a small subpopulation of cells (less than 2%) to rIL-2 was their adherence to the plastic surface. This response was noted as soon as 2 h after addition of rIL-2. 2-h rIL-2-activated plastic adherent lymphocytes were 90-98% LGL, expressed surface markers characteristic of rat NK cells (OX8 [CD8]+, asialo GM1, laminin+, OX19 [CD5]-, R1-3B3 [CD5]-, W3/25 [CD4]-, OX39 [CD25]-, Ia-, and Ig-), and expressed very high levels of cytotoxicity against YAC-1 target cells. In addition to the above markers, plastic-adherent LGLs obtained at 24, 48, or 72 h progressively expressed Ia surface antigens, but were not phagocytic and contained less than 1% monocytes/macrophages by morphology. When 24- or 48-h plastic-adherent LGL/NK cells were cultured over 3-4 d in rIL-2, the cells expanded between 30- and 100-fold, reaching densities between 2-3 X 10(6) cells/ml. These rapidly expanding LGL/NK cells also generated very high levels of LAK activity (including lysis of fresh NK-resistant solid tumor cells), expressed a phenotype characteristic of activated rat NK/LAK cells, and incorporated [3H]TdR into DNA. This technique not only provides a novel method for the purification of LGL/NK cells for in vitro studies but also provides a means for the rapid expansion of highly purified cells with high levels of broad antitumor (LAK) cytotoxicity.

The generation of natural killer (NK) cells from NK precursor cells in rat long-term bone marrow cultures.

In this report, we describe a novel long-term bone marrow culture (LTBMC) system to study the origin and generation of natural killer (NK) cells from NK precursors. Rat bone marrow was cultured for 4 wk in RPMI 1640 with 5% fetal calf serum and 2-mercaptoethanol to allow the formation of an adherent stromal cell layer containing NK precursor cells. After addition of interleukin 2 (IL-2), the LTBMC generated high numbers (up to 100-fold expansion in 7 d) of pure 3.2.3+ large granular lymphocytes with lytic activity against NK-sensitive and -resistant tumor targets, as well as antibody-dependent cellular cytotoxicity. NK activity in LTBMC could be detected 3 d after addition of as little as 1 U/ml rIL-2, whereas lymphokine-activated killer activity was found 5 d after addition of at least 10 U/ml rIL-2. In vivo depletion and in vitro complement lysis studies showed that the NK precursor cells in LTBMC did not express the NK-associated surface markers asialo GM1 or 3.2.3. We also found that LTBMC cells did not exhibit colony growth in granulocyte/macrophage or spleen colony-forming unit assays. The generation of NK cells from NK precursors required, in addition to IL-2, a second growth/maturation factor(s), which was present in the conditioned medium of the LTBMC. This LTBMC system provides a unique in vitro model to study the development of NK cells from precursor cells, the role of the bone marrow stromal microenvironment in this development, and the lineage relationship of NK cells to other hematopoietic cells.

Lack of gene rearrangement and mRNA expression of the beta chain of the T cell receptor in spontaneous rat large granular lymphocyte leukemia lines.

Using the murine cDNA clone for the beta chain of the T cell antigen receptor, we have examined four highly cytotoxic rat large granular lymphocyte (LGL) leukemia lines for the expression of unique rearrangements and mRNA transcription of the genes coding for the T cell antigen receptor. In contrast to normal rat T cells and nine rat T cell lines, the LGL leukemia lines exhibited no detectable gene rearrangements in the beta chain locus after digestion of LGL DNA by four restriction enzymes. Northern blots containing RNA from these LGL tumor lines demonstrated a low level of aberrant or nonrearranged beta chain transcription (less than 10 copies per cell) but virtually no translatable 1.3 kilobase message. These results demonstrate that LGL leukemia lines which mediate both natural killer (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) activities do not express the beta chain of the T cell receptor. The nature of the NK cell receptor for antigen remains elusive.

Evidence of suppressor cell activity in spleens of mice bearing primary tumors induced by Moloney sarcoma virus.

Spleens from Moloney sarcoma virus (MSV) tumor-bearing C57BL/6N mice contained four times the normal number of mononuclear cells and displayed a markedly elevated "spontaneous" (mitogen-independent) DNA synthesis on a per cell basis. The number of macrophages were increased three-fold while there was a slight reduction in the percentage of T lymphocytes. The phytohemagglutinin (PHA) response on a per cell basis of spleens from tumor-bearing mice was decreased about 90% when compared with normal control mice. The primary in vitro immune response to sheep red blood cells was also suppressed to levels of less than 10% of normals. The PHA response could be restored by purification of MSV spleen cells by rayon adherence columns and by removal of phagocytic cells by an iron/magnet technique. The activity of suppressor cells in MSV spleens was demonstrated in mixtures with syngeneic normal spleen cells where a marked impairment of the PHA response was observed. Spleen cells from tumor-free nude mice and normal spleen cells treated by anti-theta serum plus guinea pig complement (C'), both totally unreactive to PHA, had no such effect. The inhibitor cell in MSV spleens was shown to be insensitive to inactivation by anti-theta plus C', but could be removed by the adherence columns and the iron/magnet technique. These data suggest that this suppressor cell is a cell of the monocyte/macrophage series. Suggestive evidence was also presented that the suppressor cells belong to a proliferating population in MSV spleens. Similar suppressor cells have been previously demonstrated in spleens of mice during a variety of immune responses. Our data show, that a tumor, although stimulating the immune system, nevertheless may be suppressive on certain immune functions through the activation of suppressor cells.

Positive self regulation of cytotoxicity in human natural killer cells by production of interferon upon exposure to influenza and herpes viruses.

Augmentation of natural killer (NK) activity by influenza A/PC and HSV-1 viruses appears to be caused by the induction of interferon (IFN) within the NK cell population itself. These viruses induced high levels of IFN production by human large granular lymphocytes (LGL) that could be readily isolated from peripheral blood by Percoll density gradients. These LGL, which have been previously shown to account for and to be highly associated with endogenous NK activity, became augmented in their lytic function during the 18-h period that IFN was induced. Non-LGL helper cells did not appear to be required in the NK-IFN system (either T cells, B cells, or monocytes). Removal of these latter cell types by treatment with OKT3 plus complement, anti-IgM plus complement, or preincubation with silica or carrageenan had no effect on the ability of LGL to respond to the viruses. Production of IFN was also detected, albeit at lower levels, from monocytes cultured for 18 h with viruses, but no cytotoxic activity was induced. On the other hand, T cells, even in the presence of monocytes, showed neither property, and longer cultures, with virus up to 4 d, still did not alter the pattern. The IFN produced by both LGL and monocytes were predominantly IFN-alpha, as assessed by neutralization assays with antisera to IFN-alpha, -beta, and -gamma. In an individual with detectable serum antibodies to influenza A/PC, however, the IFN induced in LGL appeared to be gamma, presumably because of specific recognition of the virus. These data suggest an efficient positive self-regulatory mechanism in NK cells that may be readily switched on by viruses.

Chédiak-Higashi gene in humans I. Impairment of natural-killer function.

Natural-killer (NK)-cell function was profoundly depressed in donors homozygous for the Chediak-Steinbrinck-Higashi syndrome (C-HS) gene when compared with age- and sex-matched normals. This apparent defect was not simply a result of a delayed response because little cytolysis was evident in kinetics experiments even after 24 h of incubation. NK cells from C-HS donors failed to lyse adherent (MDA, CEM, and Alab) or nonadherent (K562 and Molt-4) tumor cell lines or nontransformed human fetal fibroblasts. Therefore, the apparent C-HS defect was not a result of a shift in target selectivities. In addition, the depressed reactivity did not appear to be a result of suppressor cells or factors because: (a) enriched NK populations (nonadherent lymphocytes bearing receptors for the Fc portion of IgG) from C-HS donors were low in NK-cell function, (b) C-HS mononuclear cells did not inhibit the cytotoxicity of normal cells in coculture experiments, and (c) cells from the C-HS donors remained poorly reactive even after culture for up to 7 d. The nature of the defective NK activity in C-HS patients is not clear but may lie within the lytic mechanism rather than at the level of the recognition structure or population size because the frequency of target-binding cells was normal. In vitro NK activity could be partially restored by interferon treatment. Combined with the results presented in the following paper (4), these observations suggest that the C-HS gene causes a selective immunodeficiency disorder, mainly involving NK cells. This finding, in conjunction with the high incidence of spontaneous possibly malignant, lymphoproliferative disorders in these patients, may have important implications regarding the theory of immune surveillance mediated by NK cells.

Immunosuppressive activity of a subline of the mouse EL-4 lymphoma. Evidence for minute virus of mice causing the inhibition.

Filtered culture fluids from the early in vitro passages of a subline of the C57BL/6 mouse EL-4 lymphoma, EL-4(G-), were strongly inhibitory for BABL/c vs. C57BL/6 mixed lymphocyte cultures (MLC). The inhibitory activity could be preserved by storage at -75 degrees C or 4 degrees C, thus allowing its further characterization. The inhibitory factor was particulate (nondialyzable, sedimentable at 100,000 g for 1 h), very small (recovered after 0.10 mum filtration), sensitive to UV irradiation, but heat stable (56 degrees C, 1 h) and resistant to chloroform. It was infectious, since later, noninhibitory passages of EL-4(G-) tissue culture cells became strongly inhibitory upon inoculation with the culture fluid. This data was consistent with the inhibitory factor being an infectious virus. Virus analysis by mouse antibody production tests revealed that viruses were indeed present in EL-4(G-) ascites cells and in the culture fluid, and not in a late passage of EL-4(G-) tissue culture cells which were not inhibitory. Neutralization of the inhibitory factor was achieved by pretreatment with ascitic fluid or with the sera raised against those (EL-4(G-)-derived materials which contained viruses. Mouse reference immune sera against minute virus of mice (MVM) completely neutralized the inhibitory factor in the culture fluid or in EL-4(G-) ascites cells. Two prototype MVM strains, and one Kilham rat virus preparation, did not inhibit the mouse MLC. Thus, the possibility exists that a variant of MVM, or an unidentified virus, has been grown and selected for in EL-4(G-) cells and recognized, due to its immunosuppressive characteristics. In any event, immunosuppression by EL-4(G-) cells was not mediated by the tumor cells, their metabolic products, or associated endogenous type C viruses, but by an exogenous virus, most likely a variant MVM with immunosuppressive characteristics. This adds weight to a parallel observation from our laboratory on the immunosuppressive effects of Kilham rat virus in rat lymphocyte cultures.

Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells.

To study the cellular structures involved in NK and lymphokine-activated killer (LAK) cell function, we have produced a panel of mAbs that modulate the cytolytic function of a population of cells with LAK activity that derive from large granular lymphocyte (LGL)/NK cells (adherent LAK [A-LAK] cells). In this report, we describe an mAb (3.2.3; IgG1k) that recognizes a triggering structure that is expressed on rat LGL/NK cells and A-LAK cells. This epitope is also expressed on polymorphonuclear leukocytes (PMN). The expression of the epitope identified by mAb 3.2.3 increased progressively on A-LAK cells after culture in the presence of rIL-2. mAb 3.2.3 enhanced the cytolytic activity of NK and A-LAK cells against FcR+ target cells, but not FcR- target cells. However, this effect was not induced by F(ab')2 fragments of 3.2.3. This antibody also induced the release of N-alpha-benzyloxycarbonyl-L-lysine thiobenzy esteresterase by A-LAK cells. These data suggest that the epitope identified by mAb 3.2.3 is on a triggering structure expressed on rat NK cells and A-LAK cells. The expression of the epitope recognized by mAb 3.2.3 on LGL/NK cells and PMN suggests that this structure may be analogous to that identified by the anti-CD16 (-FcR) mAbs. However, the molecule immunoprecipitated by mAb 3.2.3 was a 60-kD dimer composed of two 30-kD chains. These data suggest that mAb 3.2.3 recognizes a unique triggering structure. As mAb 3.2.3 is the first antibody recognizing a determinant with functional significance, selectively expressed on both rat NK cells and A-LAK cells, it will be a useful tool for the study of NK cell ontogeny and function, and the development of cells with LAK activity from the NK cell compartment.

Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.

Natural killer (NK) activity in the rat and human has been attributed to cells having the morphology of large granular lymphocytes (LGL). However, this association has been less clear in the mouse, largely because of difficulties in obtaining highly enriched populations of LGL from normal spleen and blood. We have previously observed that the administration of the biological response modifier (BRM) maleic anhydride divinyl ether (MVE-2) strongly augmented NK activity in lung and liver, and the augmented NK activity coincided with increased resistance to the formation of experimental metastases in these organs. The degree of NK augmentation was most striking in the liver, an unexpected and previously unreported observation. In the present study, both MVE-2 or Corynebacterium parvum induced a dramatic augmentation of liver NK activity, which reached maximum levels 3-5 d after treatment. This augmentation of NK activity in the liver coincided with a large increase in the number of lymphoid cells with the morphological characteristics of LGL that could be isolated from enzymatically digested suspensions of perfused liver. The yield of LGL per liver following BRM treatment corresponded to a 10-50-fold increase as compared to normal mice. LGL were purified from these enzymatically digested suspensions of perfused liver by depletion of adherent cells on nylon wool columns and subsequent enrichment for low-density lymphoid cells by fractionation on Percoll density gradients. The enrichment of LGL correlated with greatly increased NK activity against YAC-1. Conversely, the higher-density fractions were depleted of both LGL and NK activity. This increase in NK activity in the liver was suppressed by in vivo treatment with anti-asialo GM1 (asGM1) serum. This treatment also resulted in a corresponding reduction in both the total number and percentage of LGL. By flow cytometry analysis, the phenotype of the majority of these highly cytolytic LGL isolated from the livers of BRM-treated mice were asGM1+, Thy-1+, Ly-5+, Qa-5+, Mac-1+, and Gma-1+, whereas these LGL were Ly-1-, Lyt-2-, L3T4-, and surface Ig-. We conclude that the livers of BRM-treated mice can provide a rich source of highly active mouse LGL that could be used for further characterization of this lymphocyte subset. Further, these studies imply a potential for BRM therapy of neoplastic or viral diseases through augmentation of organ-associated immune responses.

Accumulation of adoptively transferred adherent, lymphokine-activated killer cells in murine metastases.

While close contact between lymphokine-activated killer (LAK)/adherent, lymphokine-activated killer (A-LAK) cells and tumor cells is believed to be a prerequisite for initiating the events leading to tumor cell lysis, clear evidence for the ability of these effector cells to infiltrate tumors or tumor metastases in vivo still has to be obtained. In the present study, we report that a significant fraction of adoptively transferred A-LAK cells, labeled with fluorochromes for identification, accumulates in lung and liver metastases of the B16 melanoma, the MCA 102 sarcoma and the Lewis lung carcinoma lines. Thus, 5- to 10-fold higher numbers of A-LAK cells were found in the malignant lesions compared to the surrounding normal tissue. The infiltration seemed very heterogeneous after intravenous injection of moderate numbers of A-LAK cells (15 x 10(6)). However, after adoptive transfer of 45 million A-LAK cells, an A-LAK cell/tumor cell ratio higher than 1:1 in most metastases was observed. Surprisingly, approximately 5% of the lung metastases seemed totally resistant to infiltration even though neighboring metastases were highly infiltrated. While substantial infiltration of lung metastases was seen after i.v. injection, significant infiltration of liver metastases was seen only after intraportal injection of the A-LAK cells indicating impaired traffic of intravenous injected A-LAK cells through the lung capillaries. These results present direct evidence that A-LAK cells, upon a proper route of administration, have the potential to migrate to and heavily infiltrate metastases from murine tumors of different origin.

Chédiak-Higashi gene in humans. II. The selectivity of the defect in natural-killer and antibody-dependent cell-mediated cytotoxicity function.

Antibody-dependent cell-mediated cytolysis (ADCC) of human tumor cells by FcR(+) nonadherent effector lymphocytes as well as natural killer (NK) activity was markedly impaired in Chediak-Steinbrinck-Higashi Syndrome (C-HS) patients. Compared to a more than 400-fold defect in NK activity in terms of lytic units, the abnormal ADCC response in C-HS donors was 24-fold below normal suggesting a partial but not complete overlap of lymphocytes or lytic mechanisms responsible for ADCC and NK. The ADCC mechanism against erythrocyte targets, however, was normal, thereby suggesting a qualitative difference in these two forms of ADCC. Other effector-cell functions against tumor-cell targets were normal as measured by (a) spontaneous cytolysis mediated by monocytes, (b) spontaneous cytostasis mediated by neutrophils, and (c) lectin-dependent cytolysis mediated by neutrophils. Although one C-HS patient was low in lectin-dependent cytolysis mediated by lymphocytes, the other C-HS patient was normal, thereby suggesting that cytolytic T function was not linked to the NK-ADCC defect. In addition, the proliferative response to T-dependent mitogens was also relatively normal. These results, combined with other studies showing normal cell-mediated and humoral immunity in these same patients, suggest that patients with C-HS have an immunodeficiency which is selective for NK and ADCC activity.

Natural killer cells: their roles in defenses against disease.

Natural killer cells are a recently discovered subpopulation of lymphoid cells that are present in most normal individuals of a range of mammalian and avian species. Natural killer cells have spontaneous cytolytic activity against a variety of tumor cells and some normal cells, and their reactivity can be rapidly augmented by interferon. They have characteristics distinct from other types of lymphoid cells and are closely associated with large granular lymphocytes, which comprise about 5 percent of blood or splenic leukocytes. There is increasing evidence that natural killer cells, with the ability to mediate natural resistance against tumors in vivo, certain virus and other microbial diseases, and bone marrow transplants, may play an important role in immune surveillance.

The role of natural killer cells in immune surveillance of cancer.

In the past year, a subset of natural killer cells designated 'A-NK cells' has been characterized. These immune cells appear to be able to enter solid tissues, migrate to sites of metastasis and eliminate malignant tissue cells, but spare normal tissue cells. They appear to be ideal surveillance cells, readily capable of upregulating antitumor functions in response to local activation signals.

Natural cytotoxicity of mouse, rat, and human lymphocytes against heterologous target cells.

Natural cell-mediated cytotoxicity by a particular subpopulation of lymphocytes [designated natural killer (NK) cells] in NIH Swiss nude and CBA/N mice, WF rats, and humans was demonstrated against tumor cells in 4-hour 51Cr release assays. In most studies, only reactivity against target cells of the homologous species was examined. In the present studies, mouse NK activity also was found against a rat lymphoma tissue culture cell line and against human tissue culture lines. Rat NK cells reacted not only against syngeneic tumor cells but also against heterologous tumor cell lines. In contrast to the heterologous NK activity in rodents, no significant NK activity of human peripheral blood lymphocytes against heterologous targets was found in the present studies. In mice and rats the effector cells that mediated the cytotoxicity against heterologous target cells were indistinguishable from NK cells, the effector cells being nonadherent and nonphagocytic. In addition, the mouse effector cells for heterologous activity as well as mouse NK cells were sensitive to repeated treatment with anti-Thy 1.2 serum plus complement. The specificities of these reactions were indicated by a cold target inhibition assay. The results indicated a sharing of specificities between homologous and heterologous tumor cells recognized by mouse and rat NK cells. In contrast, only the human cell lines were able to appreciably inhibit the cytotoxicity of human peripheral blood lymphocytes.