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In recent years, adoptive cell therapy of immune effector cells, such as chimeric antigen receptor-T (CAR-T) cells, natural killer (NK) cells, and epitope-specific cytotoxic T lymphocyte (CTL) cells have been employed in clinical trials. In addition, CD19 CAR-T cells have been approved by the FDA for treatment of non-Hodgkin lymphoma and diffuse large B-cell lymphoma. In this context, it is vital to detect cellular cytotoxicity and monitor the quality of ex vivo expanded immune cells before product release and patient infusion. Target cells could proliferate in parallel with effector cells during the cytotoxicity assay, making it difficult to estimate the death ratio using conventional approaches. Meanwhile, non-specific dyes or non-homogeneous biomarkers for target cells may interfere with the final readout post addition of effector cells. Here, we modified a component of the coincubation medium to suppress the spontaneous release of bis(acetoxymethyl)2,2':6',2″-terpyridine-6,6″-dicarboxylate and sustained the window at a stable range (~70%). Further, the optimized Eu-TDA method presented reliable outcomes compared with lactate dehydrogenase detection and was compatible with cytotoxicity tests for NK cells and specific CTLs. Finally, the reported assay can accurately detect death of target cells depending on the amount of hydrophilic complex and can be reliably applied in quality control and cell activity evaluation tests on co-suspended effector and target cells. SUMMARY: A medium component for cellular coincubations (and associated protocols) have been optimized and validated for cytotoxicity assays, which can reliably evaluate the potency of engineered CD19 CAR-T cells, NK cells, and specific CTLs. In particular, the reported method can be applied widely in routine assays for bi-suspended effector and target cells with a stable window., (Copyright © 2021. Published by Elsevier B.V.)

Chimeric antigen receptor (CAR)-engineered T cell therapies have been recognized as powerful strategies in cancer immunotherapy; however, the clinical application of CAR-T is currently constrained by severe adverse effects in patients, caused by excessive cytotoxic activity and poor T cell control. Herein, we harnessed a dietary molecule resveratrol (RES)-responsive transactivator and a transrepressor to develop a repressible transgene expression (RES rep ) device and an inducible transgene expression (RES ind ) device, respectively. After optimization, these tools enabled the control of CAR expression and CAR-mediated antitumor function in engineered human cells. We demonstrated that a resveratrol-repressible CAR expression (RES rep -CAR) device can effectively inhibit T cell activation upon resveratrol administration in primary T cells and a xenograft tumor mouse model. Additionally, we exhibit how a resveratrol-inducible CAR expression (RES ind -CAR) device can achieve fine-tuned and reversible control over T cell activation via a resveratrol-titratable mechanism. Furthermore, our results revealed that the presence of RES can activate RES ind -CAR T cells with strong anticancer cytotoxicity against cells in vitro and in vivo. Our study demonstrates the utility of RES rep and RES ind devices as effective tools for transgene expression and illustrates the potential of RES rep -CAR and RES ind -CAR devices to enhance patient safety in precision cancer immunotherapies., Competing Interests: Competing interest statement: H.Y., L.Y., and J.Y. are inventors of patent applications (Chinese patent application no. 202110671259.9) submitted by East China Normal University that covers the RESrep and RESind devices., (Copyright © 2021 the Author(s). Published by PNAS.)

Natural killer (NK) cells play an important role in anti-tumor immunity. Interleukin (IL)-18 is an immunoregulatory cytokine that induces potent NK cell-dependent anti-tumor responses when administrated with other cytokines. In this study, we explored the effects of combining IL-18 and IL-2 on NK cytotoxicity as well as expression levels of the NK cell receptor NKG2D in vitro. Freshly isolated PBMCs were incubated for 48 h with IL-18 and IL-2, then CD107a expression on CD3-CD56+ NK cells was determined by three-colour flow cytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human colon carcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on NK cells. The combined use of IL-18 and IL-2 significantly increased CD107a expression on NK cells compared with using IL-18 or IL-2 alone, suggesting that the combination of these two cytokines exerted synergistic enhancement of NK cytotoxicity. IL-18 also enhanced NKG2D expression on NK cells when administered with IL-2. In addition, blockade of NKG2D signaling with NKG2D-blocking antibody attenuated the up-regulatory effect of combining IL-18 and IL-2 on NK cytolysis. Our data revealed that IL-18 synergized with IL-2 to dramatically enhance the cytolytic activity of human NK cells in a NKG2D-dependent manner. The results appear encouraging for the use of combined IL-18 and IL-2 in tumor immunotherapy.

The suppressive functions of HLA-G to various immune cells have been well established. The proportion of HLA-G expression in malignant lesion cells was found from negative to 100%. However, effects for the different proportion of HLA-G expression on the cytolysis of NK cells remain to be explored. In this study, NK cytolysis to the various proportion of HLA-G1 expression on leukemia cell line K562 was investigated. Analysis of NK cell cytotoxicity was by detecting the NK cell surface CD107a expression. Data showed that NK cell cytolysis could be inhibited by the HLA-G1 expression and in a manner of HLA-G1 expression proportion dependent manner (r = 0.925, p = 0.008). Our study provided further understanding for the roles of HLA-G1 expression in malignant cell immune escaping from NK cells., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The objective of the present study was to investigate possible changes in granulysin (GNLY)-mediated cytotoxicity of peripheral blood lymphocytes in psoriatic arthritis (PsA) patients with respect to different phases of the disease. We prospectively enrolled 25 PsA patients in the active phase, 26 PsA patients in remission and 24 healthy controls. The simultaneous detection of intracellular GNLY and cell surface antigens (CD3 and CD56) was performed with flow cytometry. GNLY apoptotic protein was visualised by immunocytochemistry. Natural killer (NK) cell cytotoxicity was analysed with a cytotoxicity assay against human erythroleukaemia K-562 cells. The percentage of GNLY(+) cells did not differ significantly between PsA patients in the acute phase and those in remission; however, it was always higher than in healthy examinees due to the increased percentage of GNLY(+) cells within T cells, NKT cells, and both, and in the CD56(+dim) and CD56(+bright) NK subsets. The mean fluorescence intensity for GNLY was higher in all lymphocyte subpopulations in the acute phase than in remission and in healthy controls. Accordingly, GNLY-mediated NK cell cytotoxicity against K-562 cells of active phase PsA patients was significantly higher than that in patients in remission or in healthy controls. These findings demonstrated the involvement of GNLY in the worsening of PsA and suggested that GNLY mediated the development of joint lesions.

Multidrug resistance (MDR) confers resistance to anticancer drugs and reduces therapeutic efficiency. It is often characterized by the expression of the MDR1 gene product P-glycoprotein (or gp170) at the membrane of tumor cells. To further propose a potential complementary tool in cancer treatment, the sensitivity of gp170 tumor cells to NK-dependent lysis was investigated. Two kinds of cells were generated from wild-type K562 erythroleukemic cells: the first were derived from Taxol-selected cells and cloned, whereas the second were retrovirally transduced by the cDNA of the MDR1 gene. The last process was also applied to the human embryonal carcinoma cells called Tera-2 cells. First, both cloned and MDR-1 K562 cells appeared highly susceptible to naive NK cell killing. Interestingly, in addition, Tera-2 cells that were not sensitive to NK lysis could be killed when they expressed gp170 at their membranes. In previous data, we demonstrated that NK cell release of bimolecular complexes composed of perforin and platelet-activating factor (PAF) interacting with the PAF-R, which has to be expressed on the target cell membranes, were components of NK tumor cell killing. In the present study, we show that gp170 has the capacity to drive constitutive PAF-R expression on tumor cells, which could be responsible for hypersensitivity to NK lysis and accelerated cell death.

Human sera have shown antitumor effects mediated by tumor-specific immunoglobulin M (IgM) antibodies. Most people who have cytotoxic serum are in good health and show no evidence of exposure to tumor antigens. We characterized the serum of a healthy female adult that was highly lytic to a neuroblastoma cell line via IgM-activated complement (>60% of malignant cells were killed during the 60-min assay). Complement-dependent lysis was not mediated by other classes of serum antibodies (data not shown) which is consistent with the findings of Ollert et al. To identify the target antigen on neuroblastoma cells, we fractionated neuroblastoma cell lysates by ion-exchange chromatography. In the fraction that showed maximal IgM binding, the dominant protein was identified as the 47-kDa translational elongation factor 1alpha (eEF1alpha). We used the donor's B-cells to create hybridomas producing the antibody (B12.6.22) that bound to neuroblastoma cells and mediated cytotoxicity. This antibody recognized eEF1alpha in a specific manner. Sequence analysis of the heavy chain of B12.6.22 showed usage of VH3-23 and JH6 gene segments, with no somatic mutation. The structural similarity of B12.6.22 to antibodies of the innate immune system supports the assumption that natural antibodies are a potential source of therapeutic antibodies.

The influence of the number of differentiating agents on sensitivity of human erythroleukemic cells K562 to human leukocyte-mediated non-MHC-restricted lysis was studied. It has been shown that a 4-day treatment of cells K562 by dexamethasone (1 microM) or phorbol-12-myristate-13-acetate (100 nM) leads to a significant decrease in sensitivity of the treated cells to non-specific lysis mediated by human leukocytes. On the contrary, the treatment of cells K562 by a combination of dexamethasone and thymidine (2 mM) leads to an increased sensitivity of the treated cancer cells to non-specific lysis mediated by the above effector cells, compared with the situation when these cells were treated by dexamethasone only. The treatment of cells K562 by a combination of thymidine and phorbol-12-myristate-13-acetate demonstrates a tendency (P < 0.1) to increase the sensitivity to non-specific lysis mediated by human leukocytes, as compared with the cases, when these cells were treated by phorbol ester only. It has been shown that the changes in K562 cell sensitivity to lytic action of leukocytes, under the chosen incubation time and doses of the used agents, well compare with the changes of erythroid differentiation of the cancer cells in the same conditions.

There are few studies on cell-mediated cytotoxicity in human Chagas' disease. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) cytotoxicity activity from chagasic patients with different clinical forms of disease. To verify the cytotoxic response, we performed cell lysis assays using 51Cr-labelled K562 cells as targets. Results are reported as lytic units (LU = number of cells required for 30% lysis) per million PBMC. Exposure of patients' PBMC to Trypanosoma cruzi antigen led to an increase in cytotoxic activity compared with unstimulated patient cells against K562. Asymptomatic cardiomyopathy patients had higher responses (37.8 +/- 5.0 LU/10(6) PBMC; mean +/- s.d.) than indeterminate (11.5 +/- 3.6 LU/10(6) and symptomatic cardiomyopathy (7.8 +/- 2.5 LU/10(6)). Normal control PBMC stimulated with T. cruzi antigen had 4.36 +/- 1.31 LU/10(6)) PBMC against K562. Addition of recombinant interferon-gamma (IFN-gamma) did not lead to significant increase in cytotoxicity in any group of patients. On the other hand, recombinant human IL-12 significantly increased cytotoxic responses from symptomatic cardiomyopathy patients and normal controls who presented low levels of cytotoxicity induced by T. cruzi antigen. The combined use of IL-12 and a neutralizing anti-IFN-gamma antibody did not change IL-12-induced cytotoxic responses, showing the direct role of this cytokine on natural killer (NK) cells. NK cells were the main cells responsible for the lysis of K562 target cells as evidenced by testing cell subsets following magnetic cell sorting. These data demonstrate that chagasic patients with different clinical forms of disease have PBMC which respond to T. cruzi antigen with a cytotoxic response, and this response is up-regulated by IL-12.

A key cytolytic mediator used by killer lymphocytes, perforin (also known as pore-forming protein or cytolysin), has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis of target cells. Although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequenced, information concerning the domain structure of the perforin molecule has remained scarce. To overcome the difficulty in obtaining sufficient amounts of perforin and to further assess the functional relevance of the N-terminal portion of the perforin molecule in its lytic activity, we have attempted in the present study to produce recombinant perforins. Three forms of recombinant mouse perforin, a full-length form and two N-terminal truncated forms, have been expressed in insect (Spodoptera frugiperda (Sf9)) cells using recombinant baculovirus. Biochemical and functional characterization showed the purified full-length recombinant perforin to be capable of lysing target cells, inducing Ca2+ influx into target cells, and forming structural pores in target membranes. Significant lytic activities were also detected for the two truncated recombinant perforins lacking, respectively, the N-terminal 21 amino acid residues and 121 amino acid residues. Time course study showed that the latter acted less efficiently than the former. These results suggest the N-terminal portion of the perforin molecule to be an important, but not the only, domain responsible for the lytic function of the perforin molecule.

Earthworm coelomocytes have been used as effector cells against the human tumor target, K562. To first assess the viability of effectors, incorporation of [3H]-thymidine was tested and was higher in autogeneic (A<==A, self) than in allogeneic (A<==>B, nonself) coelomocytes. A<==>A showed significantly greater numbers in S, G2, or M phases than A<==>B coelomocytes. When A<==>A or A<==>B were cultured, no significant cell killing occurred in either, as measured in a 4-hr 51Cr release assay. A<==>A but not A<==>B killed K562 target cells. Cytotoxicity was dependent upon membrane binding between small, electron-dense coelomocytes and targets; it was enhanced by adding PHA. The heat labile supernatant from A<==>A but not from A<==>B killed K562 targets after cultivation for 10 min at 22 degrees C, but not immediately after washing. Recognition of, binding to, and killing of foreign cells in a natural killer cell-like reaction may reflect natural immunity in earthworms.

The complement membrane attack complex (MAC), the cytolytic granule protein of cytotoxic lymphocytes perforin, the streptococcal exotoxin streptolysin O (SLO), and the bee venom polypeptide melittin utilize a similar mechanism to incorporate into cell membranes, induce a Ca2+ influx and a rise in intracellular Ca2+ concentration, and produce cell lysis. At sublytic concentrations, these proteins trigger several cellular activities, including protein phosphorylation and synthesis. We have recently demonstrated that human leukemic cells treated with sublytic doses of human complement become more resistant to lytic complement doses. The study has now been extended to include three other pore-formers: murine perforin, SLO and melittin. As shown here, sublytic MAC induces in the K562 human erythroleukemic cells protection from lytic perforin, and vice versa, sublytic perforin induces protection from complement. Also, sublytic SLO and melittin increase resistance of K562 cells to lytic complement and perforin doses. The capacity of Ca2+ ionophores to induce resistance to the lytic proteins has been examined. Exposure of K562 cells to sublytic concentrations of ionomycin or A23187 for 1 h at 37 degrees C confers on them resistance to complement- and perforin-mediated lysis. The protective effects of the ionophores can be abrogated by chelation of extracellular Ca2+ and by inhibition of RNA or protein synthesis in the cells. These results indicate the following: 1) nucleated cells exposed to sublytic complement MAC, perforin, SLO, or melittin may become resistant to the four pore-formers. Physiologically, this may be regarded as an immunologic tachyphylaxis. 2) Ca2+ influx induced by these pore-formers is an essential and sufficient factor to produce this tachyphylaxis.

We have previously shown that among normal leukocytes, CD56+ and CD8+ cells express relatively high levels of P-glycoprotein (P-gp), a transmembrane efflux pump. While the physiologic significance of P-gp expression in leukocytes is unknown, the relatively high levels of P-gp in CD56+ and CD8+ cells suggest that P-gp may function in cell-mediated cytolysis. To explore this possibility we examined the effect of four inhibitors of P-gp efflux [(R)-verapamil (R-ver), (S)-verapamil (S-ver), cyclosporine A (CsA), and PSC833 (PSC)] on both the inhibition of natural killer cell (NK) function and on P-gp efflux. NK function was assayed by measuring the lysis of 51Cr-labeled K562 target cells in the presence and absence of inhibitors. All four P-gp efflux inhibitors inhibited NK-mediated cytolysis in a dose-dependent manner. The stereoisomers of verapamil were more potent inhibitors of cell-mediated cytolysis than the cyclosporines CsA and PSC. In contrast, CsA and PSC were more potent as inhibitors of P-gp-mediated rhodamine 123 dye efflux than the verapamil isomers. Both CsA and PSC maximally inhibited P-gp efflux at 3 microM, but only minimally inhibited cell-mediated cytolysis. The verapamil compounds demonstrated closer correlation between efflux inhibition of NK-mediated cytolysis. The data support a role for P-gp in NK-mediated cytolysis; however, these studies also suggest that the NK cytolytic process is multifaceted and that inhibition of the P-gp-mediated efflux mechanism only partially abrogates this process.

The method of testing the lytic potential of cell cytotoxicity, shown by whole-blood natural killer (NK) cells and K-cells, including the determination of the responsiveness of killers to interferon, is proposed. The method makes it possible to carry out mass survey of the population and to work with contaminated material. In this study the influence of different gene engineering preparations of interferon on the activity NK cells of humans and hamadryas monkeys is determined. The individual reaction of killers to their stimulation with interferon was confirmed. The study revealed that in volunteers immunized with yellow fever vaccine a decrease in the sensitivity of NK cells was observed.

Cotreatment or pretreatment of several human myeloid cell lines (KG1, HL60, U937, THP1) with the differentiation inducer DMSO was found to potentiate the antiproliferative and cytotoxic effects of TNF. In addition, TNF-resistant monocytic cell lines could be sensitized to TNF cytotoxicity by DMSO treatment. Other highly polar molecules, known to be potent differentiation inducers, showed similar effects to those of DMSO. The potentiating effect of DMSO was related neither to an up-regulation of TNF receptor expression nor to an alteration in the rate of TNF internalization and degradation. We present evidence that the TNF activities are p55 TNF receptor-mediated and are not due to insertion of TNF into lipid bilayers, an effect that could be susceptible to DMSO, as this component has been described to modify cell membrane characteristics. DMSO-induced potentiation of TNF cytostasis/cytotoxicity was restricted to myeloid leukemia cell lines. In non-myeloid cells such as fibrosarcomas, myosarcomas, thymomas, or carcinomas, DMSO was found either not to alter or to inhibit TNF-induced cell death. The latter results are in good agreement with data reported by others who suggested that DMSO could act as a scavenger of TNF-induced toxic radical formation. The potential correlation in myeloid cells between DMSO-induced changes in the cells' differentiation status and DMSO-enhanced TNF-susceptibility is discussed.

Prior study has revealed that the expression of certain major histocompatibility complex (MHC) class I molecules on the membranes of normal or transformed cells can prevent natural killer (NK)-cell-mediated killing. This has been explained by either (1) the target interference model, or (2) the effector inhibition model. In both cases, MHC class I molecules are concerned but the precise mechanisms are still unclear. The erythroleukaemia K562 cell is known as a NK-sensitive target. This sensitivity has been explained mostly as being due to lack of MHC class I antigens on the K562 membrane. However, several recent studies have indicated that the expression of MHC class I antigens on the cell does not solely explain the protection against NK cells. To elucidate the mechanism of NK-cell-mediated killing, we investigated the killing profiles of the K562 cells by NK cells. Previous studies indicated that the NK-sensitive K562 cells can express some MHC class I antigens on their surface, and become protective against NK-cell killing after treatment with human interferon-gamma (hIFN-gamma). In the present study, we show that this resistance is not rendered by MHC class I expression, because addition of anti-MHC class I monoclonal antibodies (mAb) in the killing assay of hIFN-gamma-treated K562 cells, which express MHC class I antigens as the target, did not restore the sensitivity to NK cells. Moreover, we show that spontaneously occurring K562 clones which could not express MHC class I antigens even after hIFN-gamma treatment, could be protected after treatment with the cytokine. Taken together, these results strongly suggest that the susceptibility of K562 cells to NK cell killing is due to some elements distinct from those related to MHC class I antigens.

The purpose of this study was to determine the influence of recombinant human interleukin-4 (IL-4) on the regulation of lymphokine-activated killer (LAK) and mixed lymphocyte culture cytotoxic activity. Lymphocytes from several lymphoid tissues were studied including human peripheral blood leukocytes (PBL), spleen cells, thymocytes, and thoracic duct lymphocytes. Cells were cultured with IL-4 in the presence or absence of recombinant human interleukin-2 (IL-2) in 4-day cultures. LAK and natural killer (NK) activities were measured in a standard chromium release cytotoxicity assay against LAK-sensitive, NK-resistant M14 melanoma targets and NK-sensitive K562 erythroleukemic cells. IL-4 alone does not increase NK or LAK activity under the conditions studied. However, IL-4 does inhibit the induction of cytotoxic activity by IL-2. IL-4 inhibits IL-2-induced thymidine incorporation in 3-day PBL cultures, suggesting that the inhibition of cytotoxicity is not a dilution effect due to the proliferation of noncytotoxic cell populations. IL-4 also inhibits the development of LAK and NK-like activities generated in mixed lymphocyte culture (MLC) while augmenting MLC-generated allospecific cytotoxic T lymphocyte activity. Thus, IL-4 appears to inhibit the induction of nonspecific cytotoxic effectors while augmenting the generation of MHC-specific responses. This confirms an important regulatory function for this lymphokine in the generation of cytotoxic effectors.

Freshly isolated human blood monocytes were spontaneously cytotoxic toward K562 tumor cells. During culture of the monocytes in vitro cytotoxicity decreased during the first 48 h but tumoricidal competence was restored after 3 to 4 days in vitro. These changes were accompanied by changes in both reactive oxygen intermediate generating capacity and reactive nitrogen intermediate production. Lucigenin-dependent chemiluminescence stimulated with either FMLP or PMA declined during the first 2 days in culture and was negligible during the later days in culture. Superoxide radical production in response to either FMLP or PMA remained fairly constant for the first few days in vitro and then declined. NO2- concentration in monocyte-conditioned medium was fairly constant during the first few days in vitro but increased after 6 days. The return to tumoricidal competence after 3 to 4 days in culture was decreased by the addition of NG-monomethyl-L-arginine. These results indicate that reactive oxygen intermediates are employed by monocytes in the killing of tumor cells. However, after maturation of monocytes to macrophages, this mechanism becomes less important and reactive nitrogen intermediates are employed in mediating macrophage cytotoxicity.

Target molecules for NK cells are unknown. Numerous studies have proposed putative target molecules, but have examined their role in the modulation of sensitivity to NK-mediated lysis one independently of each other. We examined the simultaneous expression of various surface molecules and the susceptibility of K562 cells to NK attack. We have previously shown that adriamycin (40 nM) and aclacinomycin (15 nM) can induce, in vitro, an increase of glycophorin A (GPA) on K562 cells, a modulation of transferrin receptor (TfR) and CD15 antigen expression and a significant resistance of cells to NK-mediated lysis. In the present work, Fc gamma receptor II (CD32) expression at the K562 cell membrane was clearly decreased after aclacinomycin-treatment but was unaltered by adriamycin-treatment. Four K562 cell clones were studied. Two clones (F and G) expressed a higher level of CD32 at the membrane (62% and 70% of erythrocyte antibody (EA) rosettes respectively) and two clones (9 and 19) expressed lower a level (18% and 7% EA rosettes respectively) than the original population (43%). The sensitivity to lysis by NK cells was increased in clones F, G and 9 but decreased in clone 19 (without alteration in the binding capacity). Relationships between the sensitivity to NK attack and the levels of simultaneous expression of CD32, TfR, CD15, glycophorin A (GPA) and MHC class I monomorphic antigens were studied. In addition, the presence at the membrane of some cellular adhesion molecules (CD54, CD58, CD29, CD18, CD56) was examined in anthracyclin-treated cells and in the four clones. The difference in the sensitivity of target cells to NK attack is not strictly related to variation of one or other of these molecules. Our previous and present data suggest that the resistance of K562 cells to NK cells may correlate with the level of erythroid maturation at the cell membrane, involving simultaneous variations in expression of several molecules such as a decrease of TfR, CD15 and CD32 and an increase of GPA.

The T24 transitional bladder carcinoma cell line, the Daudi Burkitt lymphoma cell line and the K562 erythroleukemia cell line have all been used as target cells in 51Cr release assays to measure the in vivo induced lymphokine-activated killer (LAK) cell cytotoxicity during interleukin-2 (IL-2) therapy of cancer patients. However, different relationships between the clinical response to IL-2 treatment and the LAK cytotoxicity have been reported using these three different target cells. The purpose of the present study was to evaluate whether the LAK cytotoxicities measured against these target cells represent similar effector-to-target-cell interactions, so similar conclusions may be drawn of 51Cr release assay results in which the cell lines are used as target cells. The cytotoxicity of peripheral blood mononuclear cells (PBMC) and PBMC depleted of different natural killer and T cell subsets was measured against the three targets. LAK cell recognition of targets was evaluated by cold target inhibition experiments, and the development of LAK-cell-mediated lysis with time was evaluated in 51Cr release assays of varying duration. This study shows that LAK-mediated lysis of T24 and Daudi cells was closely related and LAK cytotoxicity measured in 51Cr release assays against these two target cells may be measurement of similar effector-to-target cell interactions.

Lymphokine activated killer (LAK) activity was induced in human peripheral mononuclear blood cells by human recombinant interleukin-2. Monocytes were required for optimal rapid proliferation of cells with LAK activity. They had no influence on the expression of tumoricidal activity by the LAK cells. The effector cells killed K562 erythroleukemia cells and WiDr colon cells differently, i.e. contact areas with WiDr cells were limited, whereas the contact areas between effector cells and K562 cells were much longer. Using mixtures of hot and cold target cells it was shown that effector cells preferably bind with K562 cells, impeding the binding of WiDr cells. Differences in expression of cytotoxicity of LAK cells against WiDr and K562 cells respectively was also observed after culturing the LAK cells for a relatively longer period. Cytotoxicity against WiDr was maximal at 3-16 days after starting LAK cell generation, whereas cytotoxicity against K562 was kept constantly high for at least 21 days. The addition of biological response modifiers [PHA and anti-CD3 antibody (OKT3)] during the LAK cell induction also had different effects on the expression of LAK activity against WiDr and K562 cells. Whereas PHA, in combination with rIL-2 had no significant influence on the cytotoxicity against WiDr cells, the cytotoxicity against K562 was significantly inhibited. Addition of anti-CD3 antibody diminished the cytotoxicity against WiDr target cells and had no influence on the cytotoxicity against K562 cells.

The cytotoxicity of natural killer (NK) and lymphokine activated killer (LAK) cells against two human bladder tumor cell lines (BT-A and BT-B) was investigated using a fluorometric assay by labeling tumor cell DNA with Hoechst dye No. 33342. Our results demonstrate that BT-A and BT-B cells have low sensitivity to the cytotoxic activity of mononuclear cells (MNC) and NK cells. Cytotoxicity of MNC or NK cells against both tumor cell lines is enhanced during co-culture of the effector cells with the target cells, which suggests that BT-A and BT-B cells provide the signals which could activate MNC to exert cytotoxicity. In contrast to NK cells, IL-2-generated LAK cells showed profound cytotoxicity to BT-A and BT-B within 24 h. In addition to cellular cytotoxicity to bladder tumor cells, we also tested the effect of recombinant interleukin 1 beta (rIL-1 beta), recombinant tumor necrosis factor (rTNF), and the supernatants of co-culture of MNC or LAK cells with bladder tumor cells. The results show no cytotoxic or growth-promoting activity of rIL-1, rTNF, or the crude culture supernatants on bladder tumor cells. We found that LAK cells, but not macrophages or NK cells, may play a major role in cellular cytotoxicity against the two bladder tumor cell lines tested. From this finding we conclude that activation of LAK cells may be one important mechanism induced by adjuvant bacillus Calmette-Guérin (BCG) therapy leading to effective prevention of urothelial bladder carcinoma reappearance.

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.

Three anti K562 monoclonal antibodies (MAb) 4.3, 4.6 and 4.8 reacting predominantly with cells of myeloid lineage, were tested for antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). MAb 4.6 (IgG3k) effectively mediated ADCC against K562 cells and fresh leukemic targets with effectors from healthy donors. However, for ADCC on chronic myeloid leukemia (CML) targets, effectors from CML patients in remission needed modulation with IL-2. All MAb showed significant CDC against peripheral blood (PB) and bone marrow (BM) cells obtained from CML patients in chronic phase, and untreated acute myeloid leukemia (AML) patients. MAb displayed no CDC against PB and BM cells from CML patients in remission and BM cells of Hodgkin's Disease (HD) patients with normal BM cellularity. In clonogenic assay, colony forming units (CFU) in the BM aspirate obtained from CML patients in chronic phase were significantly reduced by treatment with MAb and complement.

Freshly isolated human peripheral blood monocytes and in vitro monocyte-derived macrophages were infected with HSV type 1 and used as target cells in a cell-mediated cytotoxicity assay. PBMC from both HSV-immune and non-immune donors were stimulated in vitro for 5 days with UV-inactivated HSV Ag and used as effector cells. Effectors from HSV-immune donors mediated virus-specific lysis of both monocyte and macrophage targets, whereas effectors from non-immune donors failed to mediate target cell lysis. Mean virus-specific lysis of autologous monocytes was (8.5 +/- (+/- 2.0)%) compared to a threefold greater virus-specific lysis of autologous macrophages (24.7 (+/- 4.3)%). More than 70% of this lysis was mediated by CD16- T lymphocytes. Further analysis demonstrated that the majority of the lysis against autologous and allogeneic targets was HLA-DR-restricted and mediated by CD4+ CTL. However, CD8+ CTL also contributed to the lysis of autologous targets as well as allogeneic targets having a common HLA-A and/or -B determinant. The HLA-restricted cytotoxicity was virus-specific as HSV-infected, but not CMV-infected, cells were lysed. CTL-mediated lysis of HSV-infected monocytes and macrophages may be of significance in the anti-viral and immunoregulatory host response.

The cytotoxic potential of blood lymphocyters from healthy donors was tested against freshly isolated lung cancer cells and the erythroleukemia K562 cell line in short-term 51Cr release assays conducted at an effector:target ratio of 50:1. Most donors exhibited significant activity against K6-562 cells. By contrast, fresh tumor cells were refractory, only 6 of 30 showing significant cytotoxicity. The low susceptibility of these tumor cells was confirmed in third-party cold inhibition assays in which they interfered minimally with killing of K562 targets under conditions in which unlabeled K562 cells efficiently blocked cytotoxicity. Cells prepared from normal lung tissue and Raji cells also failed to inhibit killing. Although in comparison to the K562 cell line freshly isolated tumor cells were resistant, their susceptibility may not be so low as to be biologically irrelevant, inasmuch as boosting of natural killing activity by interferon induced levels of cytotoxicity against both types of target cell that were unattainable by unstimulated effectors. Interferon-boosted killers were lytic for "normal" lung cells and the Raji cell line.

The abilities of Escherichia coli-derived human interferon gamma (IFN-gamma) and E. coli-derived human interferon-alpha A (IFN-alpha A) or -alpha 2 (IFN-alpha 2) to augment natural killer (NK) cytotoxicity were compared. When low concentrations (less than 10 antiviral units/ml) of interferons were used, and equal numbers of antiviral units of E. coli-derived IFN-gamma and E. coli-derived IFN-alpha A or IFN-alpha 2 were compared for their ability to augment NK, E. coli-derived IFN-gamma was found to be more active in augmenting NK against the K562 targets, than E. coli-derived IFN-alpha A or IFN-alpha 2. Antiviral units in these experiments were determined by the standard cytopathic effect assay using vesicular stomatitis virus (VSV)-challenged human fibroblasts, trisomic for chromosome 21. However, when these interferons were compared on a weight basis (ng/ml) or on a molar basis, their ability to augment NK against the K562 targets was comparable. These differences in the relative abilities of these interferons (when their concentrations were expressed in antiviral units/ml) to augment NK, were due to an approximately 100-fold difference in their specific activities (antiviral units per mg of interferon). These were 1.8 X 10(6) units/mg for E. coli-derived IFN-gamma, 2.0 X 10(8) units/mg for E. coli-derived IFN-alpha A, and 1.8 X 10(8) units/mg for E. coli-derived IFN-alpha 2. At concentrations higher than 10 units/ml, all these interferons showed a similar ability to augment NK. Studies on the kinetics of the augmentation revealed that in vitro treatment with E. coli-derived IFN-gamma for several hours was necessary for augmentation of NK against targets from haemopoietic human tumour cell lines (K562, Daudi). In contrast, alpha interferons were able to augment NK after treatment in vitro for significantly shorter periods (30 min or less with certain donors). Augmentation of NK cytotoxicity of human peripheral blood mononuclear leucocytes by E. coli-derived IFN-gamma was not accompanied by the induction of interleukin 2 (IL-2) production, suggesting that IL-2 is not involved in the augmentation of NK by IFN-gamma. A monoclonal antibody specific for human IFN-gamma blocked augmentation of NK by E. coli-derived IFN-gamma and natural IFN-gamma, but not by E. coli-derived IFN-alpha A or staphylococcal enterotoxin A (SEA).(ABSTRACT TRUNCATED AT 400 WORDS)

IFN-gamma has been shown to reduce the sensitivity of tumor cells to lysis by NK cells. The close relationship between NK cells and lymphokine-activated killer (LAK) cells has prompted us to investigate whether IFN-gamma pre-treatment also affects the sensitivity of tumor cells to lysis by LAK. We have shown previously that IFN-gamma can induce a significant reduction in the sensitivity of both cultured and fresh (surgically obtained) human tumor cells to lysis by LAK. Herein we show that changes in the sensitivity to LAK lysis of cultured human tumor cells can be induced by as little as 1 to 10 U/ml of IFN-gamma; a dose well within the range that can be achieved in vivo. Protection is induced within hours after treatment with IFN-gamma and is dependent on the continued presence of IFN-gamma. Tumor cells cultured in IFN-gamma for several days remain less sensitive to lysis and do not become refractory to IFN-gamma-mediated protection. In the absence of IFN-gamma, treated tumor cells regain "normal" sensitivity to lysis within 48 to 72 h. We have also investigated the mechanisms by which IFN-gamma reduces tumor cell sensitivity to LAK lysis using cold target competition, monolayer depletion, direct binding, and kinetic assays. IFN-gamma pre-treatment does not alter the kinetics of tumor cell lysis by LAK. Our data are most compatible with a model in which IFN-gamma reduces the ability of a subpopulation of tumor cells to induce the LAK effector cell to initiate lysis. These results are closely parallel to observations made on the IFN-mediated protection of targets from NK lysis and support the notion that NK- and LAK-mediated lysis are closely related. These results may have significance in vivo because high levels of IFN-gamma may be present at the tumor site or may be induced after therapeutic immunomodulation.

Mouse YAC-1 and human K562 leukemic cells were treated in vitro with fibroblast interferon (IF) and tested for their susceptibility to NK effector lymphocytes. In both cases a decrease in target susceptibility was induced by the IF treatment. "Cold" competition experiments confirmed that loss or masking of NK target structures occurred in IF-pretreated cells. In fact, when radio-labeled untreated cells were used as targets, IF-pretreated leukemias produced inhibitory effects lower than those mediated by intact cells. However when IF-pretreated targets were used, cold cells either intact or preincubated with IF gave similar competitive effects. These data suggest that IF modulates differentially distinct subsets of NK target structures.

The cell believed to be responsible for human spontaneous cytotoxicity or natural killing (NK) has been described recently as a large granular lymphocyte (LGL). Because the intracytoplasmic granules of LGL appear to be peculiar to the subpopulation of mononuclear cells containing NK effectors, we have investigated their possible requirement in the cytolytic process. Experiments reported here show that Sr++, a cation known to cause degranulation of granulocytes, caused a dose- and time-dependent inhibition of the NK activity of human peripheral blood mononuclear cells. A single-cell NK assay showed that the number of cytotoxic cells was not significantly reduced, but that Sr++ inhibited NK by preventing individual effector cells from engaging in multiple lethal hits. Moreover, the inhibition of NK appeared to be reversible; culture in vitro of Sr++-treated PBMC resulted in the eventual return of cytolytic activity in these cells. Augmentation of NK by interferon (IFN) was similarly blocked by Sr++. In addition, Sr++ also inhibited the NK activity of IFN-prestimulated effector cells and, as before, NK function returned during subsequent culture. These results indicated that the return of NK function after Sr++ treatment was the result of reactivation of cytotoxic cells rather than recruitment of new effectors. We have shown in other studies that Sr++ degranulates LGL, and the data presented here provide support for the hypothesis that the intracytoplasmic granules of LGL are required for NK-mediated cytotoxicity.

Several animal models have been developed in which the adoptive transfer of specifically immune syngeneic T cells has been shown to mediate the eradication of established tumors. In adoptive chemoimmunotherapy (ACIT) of disseminated FBL leukemia with cyclophosphamide and immune T cells, the major effector T cell has been shown to be a noncytolytic Lyt-1+2- T cell that mediates its therapeutic effect without the participation of CTL. Because studies in other models have suggested that CTL can mediate an anti-tumor effect, the efficacy of Lyt-2+ T cells rendered highly cytolytic before adoptive transfer in ACIT of disseminated FBL was examined. The results demonstrated that such CTL had a detectable but limited therapeutic effect in the treatment of FBL. Because this limited activity of transferred purified CTL might have reflected a requirement for helper T cells to produce IL 2 for promotion of the in vivo survival and proliferation of the CTL, the effect of administering IL 2 to tumor-bearing hosts after transfer of CTL was examined. A dose of IL 2 previously shown to induce in vivo proliferation of transferred T cells rendered CTL that were minimally effective alone curative in ACIT of FBL leukemia. Thus, either lymphokine-producing T cells or the lymphokines produced by these cells are necessary for the full expression of the in vivo therapeutic potential of CTL.

Lymphokine-activated killer (LAK) cells are cytotoxic for a variety of autologous and allogeneic tumor cells as well as modified autologous cells. It is assumed that LAK cells lyse their targets solely by direct cell to cell contact, possibly involving the degranulation and exocytosis of pore-forming elements, similar to that observed with cytotoxic T lymphocytes and NK cells. Reported here are studies demonstrating that LAK cells release factor(s) that are cytotoxic for a human breast carcinoma cell line, MCF-7, when stimulated with tumor cells. The factor(s) are slow acting and maximum cytotoxicity is observed only in a 72-h cytotoxic assay. The ability of LAK cells to secrete cytotoxic factor(s) is dependent on both the ratio of LAK cells to stimulating tumor cells as well as the length of their coincubation. A number of similarly slow acting cytokines that are cytostatic and/or cytotoxic for tumor cells have been described. We tested the ability of specific polyclonal antibodies directed against TNF, IFN-alpha, IFN-beta, and IFN-gamma to neutralize the cytotoxic supernatant activity. Only antibodies specific for IFN-gamma and TNF were neutralizing. We measured the amounts of IFN-gamma and TNF in the cytotoxic supernatants and determined that increased amounts of IFN-gamma and TNF were released after LAK cell-tumor cell interactions compared to supernatants of LAK cells alone or tumor cell alone. Comparable concentrations of human rIFN-gamma and rTNF resulted in similar levels (50 to 90%) of MCF-7 cell cytotoxicity as those observed with the stimulated LAK cell supernatants. We thus concluded that the majority of the cytotoxic activity released by LAK cells when stimulated with tumor cells was attributed to the synergistic activities of IFN-gamma and TNF. The significance of these observations in relation to the possible mechanisms by which LAK cells mediate cytolysis is discussed.

K562 cells of different proliferative activity were tested for their capability to form lytic and non-lytic conjugates in agarose with peripheral blood mononuclear cells (MNC). Simultaneously, the conjugating MNC were analysed in suspension by monoclonal antibodies (mAb) defining subsets of T cells, natural killer (NK) cells, and monocytes (OKT 8, OKT 4, Leu 7, OKM 1, Mo 2). It is demonstrated that the pattern of conjugating (binding, lysis) is dependent upon the proliferative status of the target cell population. Target cells of intermediate division rate are optimally lysed by MNC of NK phenotype, whereas targets of high and low division rate bind cells predominantly of T cell phenotype, but are not killed. Non-cytolytic conjugating MNC are shown to inhibit significantly the cell cycle progression of their bound tumour target cells. There is evidence of an additional cytostatic effect on non-bound K562 cells in agarose dishes containing effector MNC, possibly mediated by soluble factors released from NK cells. Adherent monocytes contribute only little to cytolysis and cytostasis in our testing system. There is no correlation between the expression of transferrin receptors on target cells as determined by the OKT9 mAb and the extent of killing. Transferrin receptors may, however, be involved in the step of target cell recognition by MNC exhibiting the OKT 8+ phenotype.

Human monocytes can be activated to release a 40,000-Da cytostatic protein factor (CF). In this report we have investigated the cytolytic activity of CF on WEHI 164 cells which are sensitive to monocyte-mediated cytolysis. Monocyte supernatants containing CF induced cytolysis of murine WEHI 164 sarcoma cells, as determined in a 51Cr-release assay. Preincubation of WEHI 164 cells with actinomycin D enhanced cytolysis induced by supernatants containing CF, suggesting that CF may be involved in drug-dependent cellular cytotoxicity. The cytolytic activity was profoundly inhibited by a rabbit antiserum raised against purified CF, indicating that the cytolytic activity in the supernatants was in fact mediated by CF. These results indicate that CF may be an important effector molecule in monocyte-mediated cytostatic and cytolytic reactions.

Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) and lipoxin B (5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid), two newly isolated compounds derived from the oxygenation of arachidonic acid in human leukocytes, inhibit the cytotoxic activity of human natural killer (NK) cells. Dose-response studies showed that both lipoxin A and lipoxin B inhibit, at submicromolar concentrations (ID50 10(-7) M), NK cell activity assayed against K562 target cells. Prostaglandin E2 (PGE2) also inhibited cytotoxicity, whereas both 15-HETE (5(S)-hydroxy-5,8,11,13-eicosatetraenoic acid) and leukotriene B4 (synthetic and biologically derived) were ineffective. PGE2 stimulated a time- and dose-dependent increase in intracellular cAMP, which was accompanied by a decrease in NK target cell binding. Lipoxin A and lipoxin B did not elevate intracellular cAMP, nor did they inhibit target cell binding. Together these findings suggest that lipoxin A and lipoxin B abrogate NK cell cytotoxicity at a step distal to target effector cell recognition. In contrast, PGE2 appears to exert its effect, at least in part, on cytotoxicity indirectly by decreasing the binding between target and effector cells (in vitro). Moreover, they suggest that novel oxygenated derivatives of arachidonic acid (i.e., lipoxin A, lipoxin B) may regulate the activities of NK cells.

We generated a homogeneous population of cells with cytotoxic activity termed "adherent lymphokine-activated killer" (ALAK) cells from the peripheral blood of nine patients in the chronic phase of Ph1 positive chronic myelogenous leukemia (CML). The selective enrichment of CML ALAK cells depended on their propensity to adhere to plastic and proliferate when cultured in the presence of recombinant interleukin-2 (rIL-2) for 14 days. Culture of peripheral blood mononuclear cells under these conditions resulted in growth of a uniform population of cells with morphologic characteristics of large granular lymphocytes. The NKH1+/CD3- phenotype associated with IL-2-stimulated natural killer (NK) cells was present on 79% +/- 9% of cells. Absence of colony formation in conditions promoting the growth of CFU-GEMM indicated that the CML ALAK population was not contaminated with viable hematopoietic progenitors. Cytogenetic analysis of the CML ALAK population revealed 119/120 Ph1 negative metaphases and l/120 Ph1 positive metaphase in six patients. Southern blot analysis of CML ALAK failed to demonstrate a bcr gene rearrangement in seven patients known to have a bcr gene rearrangement in myeloid cells. Comparison of ALAK populations derived from peripheral blood of CML patients and normals revealed similar cytotoxicity against the NK-sensitive K562 cell line (104 +/- 36 LU v 88 +/- 19 LU; P = NS) and the NK-resistant Raji cell line (93 +/- 26 LU v 98 +/- 28 LU; P = NS). The proliferative capacity of CML ALAK cells (101 +/- 33 fold expansion) exceeds the growth potential of the normal ALAK cells (22.3 +/- 3 fold expansion; P = .02). Direct comparison of equal numbers of CML ALAK cells and a CML LAK cell population produced by incubation of peripheral blood mononuclear cells in rIL-2 for 14 days without adherence revealed that the CML LAK population had significantly lower lytic activity against K562 and Raji cell lines. We are able to expand CML peripheral blood mononuclear cells to provide a population of ALAK cells with potent cytotoxic activity. The CML ALAK population is relatively homogeneous, not contaminated with viable stem cells, not derived from a malignant lineage, and more cytotoxic than equal numbers of CML LAK cells. Further studies are underway to determine if this ALAK population may be effective in autologous killing of chronic myelogenous leukemia stem cells.

Expression of the transferrin receptor on target cell lines has recently been implicated as a determinant of susceptibility to cytolysis by natural killer (NK) lymphocytes. We have examined this proposed relationship in several ways. First, K562 (a cell line highly vulnerable to NK lysis) cells were grown for 24 h in the iron chelator desferrioxamine. Under these conditions, the cells doubled their surface transferrin receptor expression as determined both by radioligand binding and surface binding of the OK-T9 monoclonal anti-transferrin receptor antibody. In contrast, cells grown for the same period of time in hemin halved their receptor expression. This fourfold change in transferrin receptor expression between the desferrioxamine-treated and hemin-treated cells produced no change in susceptibility to NK cytolysis. Second, HeLa (a cell line which in its native state is very resistant to NK cytolysis) cells were compared with K562 cells with respect to surface transferrin receptor expression. The difference in NK susceptibility of the two cell lines was not reflected in differences in transferrin receptor expression: the K562 cells expressed approximately 1.5 X 10(5) receptors per cell while HeLa cells expressed 2.0 X 10(5) receptors/cell. Third, infection of HeLa cells by measles virus greatly increased their susceptibility to NK lysis but produced no change in surface transferrin receptor expression. Furthermore, when measles-infected HeLa cells were grown for 6 d in medium supplemented with iron-saturated human transferrin they underwent a 50% reduction in receptor expression but no change in NK susceptibility. Finally, possible alterations in the surface expression of NK target antigens on modified cells were further assayed by their ability to serve as cold-target inhibitors of cytolysis of NK-sensitive target cells. We examined two groups of cells in which transferrin receptor expression was reduced. These were the transferrin-treated, measles-infected HeLa cells with the 50% receptor reduction, and K562 cells grown in medium containing hemin and iron salts where the reduction was five- to sixfold relative to control. In neither case was there a change in the apparent expression of NK target antigen(s). We conclude that there is a discordance between transferrin receptor expression and susceptibility to NK cytolysis in the model systems examined. Therefore, it is unlikely that the transferrin receptor per se is the target recognition structure for human NK cells, although a role in concert with other, as yet undefined molecules, cannot be excluded.

To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.

We studied the action of alpha-interferon (IFN) and interleukin-2 (IL-2) on natural killer (NK)-rich fractions and autologous tumor cells from two patients with hairy cell leukemia (HCL). The addition of IFN or IL-2 to the NK-rich fractions resulted in a significant increase in NK activity against the autologous tumor cells. This stimulatory effect was blocked if the target hairy cells (HCs) were preincubated with either IFN or IL-2. Pretreatment of the HCs with anti-Tac antibody entirely prevented the blocking effect of IL-2 and partially the blocking effect of IFN. One patient was treated with recombinant alpha c-IFN. After 2 months there was a dramatic reduction in the number of HCs in the peripheral blood coincident with the loss of the protection effect of IFN against NK lysis of the patient's HCs. NK activity against autologous tumor cells correlated poorly with that against the K562 cell line. We conclude that there is a discordant effect of IFN and IL-2 on NK activity and HC sensitivity to lysis. The Tac receptor appears to play a role in this sensitivity. Caution should be exercised in extrapolating the effects of NK activity against K562 cells to those on HC targets.

Natural killer (NK) activity is inhibited by the contact of peripheral blood lymphocytes with primary monolayer cell cultures of both benign and malignant origin. In this study the effect of interferons (IFNs) on the inhibition has been analysed. Both alpha IFN- and gamma IFN-pretreated peripheral blood lymphocytes are effectively inhibited by monolayer target cells. IFN treatment of lymphocytes does not change cytotoxicity against the inhibitory target cells, although it enhances reactivity against NK-sensitive target cells. Treatment of monolayer cells with interferons significantly reduces their inhibitory capacity. However, diminished inhibition of NK activity by the IFN-treated target cells is not associated with increased lysis, probably due to their decreased sensitivity to natural killer cytotoxic factors (NKCF). In 18.5% of the cases studied, monolayer target cells induced endogenous IFN production in lymphocytes. In these cases no inhibition of the NK activity of the effector cells was seen. According to the results of this paper, IFNs have a dual effect on the NK regulatory system: they enhance the NK activity of the effector cells against NK-sensitive target cells, and they change the NK resistant target cells in a way that makes them less inhibitory to NK activity but simultaneously more resistant to the toxic factors secreted by NK cells.

Human peripheral neutrophils treated with recombinant human interferon-gamma (IFN) inhibited tritiated thymidine (3H-TdR) uptake by various tumor cells. The concentration-response curve of IFN required for induction of cytostatic activity of neutrophils showed two peaks. Short time incubation of neutrophils with IFN at 4 degrees C was sufficient for inducing neutrophil cytostasis. When neutrophils pretreated with IFN for 5 min at 4 degrees C were further treated with trypsin, cytostasis by the treated neutrophils was decreased depending on the concentration of trypsin, whereas cytostasis by neutrophils pretreated with IFN for 180 min at 37 degrees C was not inhibited by trypsin treatment. Cytostatic activity induced by IFN was inhibited by pretreatment of IFN with anti-IFN-monoclonal antibody.

Dimethyl sulfoxide (DMSO) treatment of the Friend erythroleukemia cell line GM 979 markedly increased its susceptibility to natural cytotoxicity by splenocytes from normal inbred DBA/2 mice. Cytotoxicity occurred with normal adherent spleen cells as well as dextran-elicited peritoneal exudate (PE) cells but not with resident PE cells. Susceptibility of the leukemia cells to natural cytotoxicity increased to maximum levels upon treatment with 210 mM DMSO for 2-3 days. The natural cytotoxicity assayed by the 51Cr release procedure was first detectable after 9 hours of incubation and reached maximum levels by 24-30 hours. Although both DMSO and n-butyric acid induced rapid erythroid cell differentiation of the GM 979 cells, and both resulted in increased hemoglobin synthesis, only DMSO treatment enhanced the susceptibility of the cells to natural cytotoxicity by normal splenocytes. Cell-free supernatants from adherent spleen cells cocultured with DMSO-treated GM 979 cells for 6-15 hours were markedly cytotoxic for cultures of other chromium-labeled DMSO-treated leukemia cells. Supernatants from cultured adherent spleen cells alone, or lysates of DMSO-treated leukemia cells, did not possess cytotoxic activity. Resident peritoneal macrophages also had no cytotoxic activity against DMSO-treated cells, and culture supernatants from resident PE cells, even after incubation with DMSO-treated target cells, failed to show significant levels of cytotoxicity. These results indicate that normal splenic adherent cells as well as elicited PE cells have the ability to lyse DMSO-treated leukemia cells.

This paper reports on the characteristics of killing by a human and a murine tuberculin (PPD)-specific T helper clone of targets to which PPD was attached via the lectin concanavalin A (Con A). The killing was specific for PPD from M. tuberculosis; and targets coupled to Con A alone or to PPD from M. paratuberculosis were not killed. Target cells carrying Con A-PPD were more effectively lysed than PPD-pulsed cells. This form of lymphocyte killing, though highly significant, was inefficient. Maximum killing of PPD carrying targets was 30-40% at effector to target ratios of 20:1 and at 16 h. Cells carrying 2 x 10(6) molecules of PPD and less than 1.5 x 10(6) molecules Con A per cell were killed most efficiently. A major distinction between this helper T cell killing and that mediated by cytotoxic T cells was that both TH clones displayed bystander lysis and killed PPD uncoupled targets when these were cultured with syngeneic PPD-bound targets. This suggests that the mechanism of cytotoxicity may involve soluble mediators.

The role of oxygen radicals in lysis of K562 target cells by human natural killer (NK) cells was determined by addition of scavengers of these free radicals. Lysis was greatly reduced under hypoxic conditions. Superoxide dismutase and cytochrome c, scavengers of superoxide anions, and catalase and scavengers of hypochlorite had no effect on lysis. Of 15 hydroxyl radical scavengers tested, 13 inhibited lysis. These were not toxic, because cell morphology and spontaneous chromium release were not affected and preculture with scavengers was not inhibitory. These scavengers differed widely in structure, but degree of inhibition of lysis correlated with their rate constants (k) for reaction with hydroxyl radical (k vs log inhibitor concentration required to decrease lysis by 50%: r = -0.9202, p less than 0.001), showing that inhibition was due to inactivation of the hydroxyl radical. Target cell binding was not reduced at concentrations that inhibited lysis. Inhibitors of the lipoxygenase pathway also decreased lysis, suggesting this pathway to be the source of hydroxyl radicals. In view of the reported requirements for hydroxyl radical-mediated lipid peroxidation for optimal secretory activity in a number of cell types, it appears that the generation of hydroxyl radicals by NK cells is required for delivery of cytotoxic factors.

Owl monkey mononuclear cells were separated from peripheral blood by centrifugation on Ficoll gradients, removal of adherent cells, and subsequent separation on discontinuous Percoll gradients. Lymphocytes recovered from the various fractions were tested for cytotoxic reactivity immediately after isolation. Low-density cells, enriched in large granular lymphocytes (LGL), demonstrated cytotoxic activity against the human natural killer-susceptible cell lines MOLT 4 and K562. In addition, IL-2-independent T-cell lines which had been obtained by immortalization with the primate herpesvirus Herpesvirus saimiri showed cytotoxicity, even after prolonged culture in vitro, similar to that demonstrated by fresh LGL. Cytotoxic activity of these lines was regulated by IL-2 in a fashion which appeared to be independent of the growth-promoting effects of this lymphokine. These results indicate a function for IL-2 beyond its role in supporting cellular proliferation. Cytotoxic activity could also be demonstrated in culture fluids from one of these cell lines (70N2). In addition, these results indicate the usefulness of immortalized cell lines (like 70N2) as a potential source for studies of the biochemical characterization and purification of supernatants containing cytotoxic factors.

The evidence that NK cells can recognize and kill allogeneic lymphocytes has hitherto been based mainly on experiments in intact animals. Here we report results from an in vitro assay, showing allogeneic lymphocyte cytotoxicity in cell suspensions enriched for NK activity against tumour cells by Percoll gradient centrifugation of nylon-wool non-adherent cells. The addition of phytohaemagglutinin (PHA) to the NK-target cell cultures greatly enhanced the cytotoxic response against K562 and allogeneic, but not syngeneic, lymphocytes. The effector cells of ALC are present in the spleen of both euthymic and athymic nude rats, and to a lesser extent in the blood. ALC is augmented by interferon pretreatment of the effector cells, and by depleting the effector cell suspensions of all T cells and helper T cells with the monoclonal antibody MRC Ox19 and W3/25, respectively. Conversely, the activity was nearly abolished by depleting the cell suspensions of MRC Ox8+ cells reacting with rat cytotoxic T cells and NK cells. Furthermore, removal of residual B cells (Ox12+ cells) from the effector cells or attempts to block any putative antibody-dependent cellular cytotoxic mechanism in vitro with the monoclonal antibody Ox12 did not inhibit the NK activity against allogeneic lymphocytes nor against tumour cells. ALC in vitro did not discriminate between T and B or large and small lymphocyte targets. These characteristics of the ALC effector cells substantiate that they are present within the thymus-independent population of cells with NK activity, and are dependent on neither B cells nor immunoglobulin for their recognition and destruction of the target.

Treatment of PBL or Percoll-isolated LGL with anti-transferrin antibodies plus complement reduced their natural killing activity against K-562 cells between 30 and 70%. The same antibodies inhibited natural cytotoxicity when added directly to the assay. Similar depletion or inhibition of NK cytotoxicity was observed when using HeLa cells as targets. The decrease or inhibition by transferrin antibodies was less marked when IFN-treated PBL or LGL as effector cells were used. The inhibition of anti-transferrin antibodies seems to be located at the level of the effector cell population. When PBL but not target K-562 cells were pretreated with anti-transferrin antibodies and were washed before use in the assay, cytotoxicity was decreased by 50%. In addition, about 80% of the LGL positively selected on anti-transferrin plates stained with Leu-11. Furthermore, no reduction by anti-transferrin antibodies plus complement treatment of PBL or LGL, or inhibition by antibodies alone, was observed when the cells were tested against HSV-1-infected cells. Membrane extracts from LGL inhibited NK cytotoxicity against K-562 or HeLa cells. Moreover, the inhibitory component of this extract was removed by anti-transferrin IgG but not by control IgG. These results are in agreement with the recent hypothesis that NK cells recognize the transferrin receptor in tumor target cells, because both the transferrin receptor and anti-transferrin antibodies may share a similar structure that interacts with the NK cells.