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Pretreatment of human large granular lymphocytes (LGL) or unseparated peripheral blood mononuclear cells with interferon (IFN) resulted in a significant augmentation of natural killer (NK) activity. This increase was paralleled by an increase in the 2'-5'A synthetase activity. In order to investigate the possibility that IFN might be inducing augmentation of NK cells via the 2'-5'A pathway, we tested the effects of nonphosphorylated core material [(A2'p)2A] and of the triphosphorylated form of the 2'-5'A [ppp(A2'p)2A]. The core material had no detectable effect on NK activity. In contrast, when experiments were performed with the triphosphorylated form of 2'-5'A, NK activity was stimulated. In order to achieve activation, permeabilization of LGL with calcium chloride was necessary and, under these conditions, a dose-dependent augmentation of NK activity was seen. However, the calcium treatment had considerable toxic effects on basal levels of NK activity. Collectively, these results suggest that IFN may be inducing augmentation of NK activity via the 2'-5'A pathway. Further studies will be necessary to determine the effects of IFN and/or 2'-5'A on subsequent activation steps in the process leading to cytotoxicity by NK cells.

Human peripheral blood lymphocytes cultured in vitro for 2 days in serum-free conditions produced a natural killer (NK) cytotoxic factor (NKCF) which selectively killed NK-susceptible targets. Optimal release of NKCF was achieved under serum-free conditions, while the presence of fetal calf serum inhibited both the production and activity of the factor. Mechanistic studies with NKCF demonstrated that the factor could be adsorbed by the target cells within 6 h, with no further exposure to NKCF required for maximal levels of lysis of the treated targets after additional 30-48 h of incubation, as assessed by a 111I release microcytotoxicity assay. NKCF adsorption to target cells and its cytotoxic activity were inhibited by some phosphorylated sugars (mannose-6PO4 and glucose-6PO4), but not by fructose-6PO4 or nonphosphorylated sugars (mannose, glucose, galactose). These results suggest a role of sugar-6PO4 at the level of interaction of NKCF with NK target cells. This was further supported by the finding that inhibition of target cell glycosylation by tunicamycin also inhibited absorption of NKCF to the target cells and direct killing by NKCF. Therefore, it appears that NKCF is a large granular lymphocyte produced factor which produces lysis as a result of the interaction with glycosylated structures on target cell membranes. Purification studies were performed to begin biochemical characterization of human NKCF. The results indicated that NKCF has an apparent molecular weight between 20,000 and 40,000 dalton. Such approaches with radiolabeled NKCF should be useful for the further study of the biochemical characteristics of human NKCF and of its mechanism of action. The ability to elicit NKCF under serum-free conditions should facilitate its testing, purification, and biochemical characterization.

We describe a method for the purification of human and rat large granular lymphocytes (LGL), which are known to be the mediators of natural killer (NK) activity in these species. Plastic non-adherent and nylon wool passed blood mononuclear cells were separated into 7 fractions by discontinuous density gradient centrifugation on Percoll. Low density cells were highly enriched in LGL (up to 85% purity), whereas high density cells were typical small and medium sized lymphocytes devoid of NK activity. Human LGL could further be enriched by depleting high affinity sheep erythrocyte rosette-forming cells from the LGL-enriched Percoll fractions (resulting in up to greater than 90% purity). One critical variable in the separation technique was osmolarity, since the separation did not work optimally, if 290 mOsmoles/kg H2O in the Percoll solution was exceeded.

Natural killer (NK) activity in rats and NK and antibody-dependent cell-mediated cytotoxicity in mice have been shown to be rapidly augmented by in vivo treatment with a variety of viruses and immune adjuvants. Poly I:C and other interferon inducers also caused boosting in NK-cell activity, with peak levels occurring at about the time of peak interferon levels. Crude and purified virus-induced interferon itself had strong boosting effects in mice, both when inoculated in vivo and when incubated briefly with normal spleen cells. The in vitro activation by interferon or by poly I:C was completely abrogated by anti-interferon. Interferon also appears to play a central role in augmentation of human NK-cell activity. Inoculation of normal volunteers with influenza virus or of a patient with poly I:C produced rapid boosting. In vitro incubation of peripheral blood mononuclear cells with various interferon preparations for 1 hour or overnight usually produced an appreciable increase in lytic activity.

Six lymphoid human interleukin-2s (nIL-2s) [four from peripheral blood mononuclear cells (PBMC) and two from JURKAT cells] and six recombinant IL-2s (rIL-2s) were obtained for comparative evaluation. The main issues addressed were possible differences among the preparations in potency in T cell growth assays and other functional assays, and the possible presence of other cytokine activities or contaminants. Each preparation was assigned a standardized IL-2 activity in reference units (RU) by comparing its T cell growth promoting activity against the Biological Response Modifiers Program IL-2 (JURKAT) reference reagent. Relative to the IL-2 unitage indicated by the suppliers, the RU varied from 110-fold less to 38.5-fold more for the various preparations. Two nIL-2s and two rIL-2s contained significant levels of endotoxin. One nIL-2 contained low levels of both alpha and gamma interferon (IFN), and one nIL-2 had a high level of gamma IFN. All other IL-2s were negative for IFN activity. All IL-2 preparations significantly augmented human natural killer (NK) activity, although the amount of RU required varied from 0.1 to 50 RU. Four nIL-2s and three rIL-2s induced human PBMC to produce gamma IFN, whereas two nIL-2s and one rIL2 did not. All nIL-2s had substantial amounts of B cell growth factor activity, whereas none of the rIL-2s consistently displayed this activity. All IL-2s stimulated the tritiated thymidine [3H]TdR incorporation of human PBMC in the absence of other stimuli, in addition to augmenting the response to mitogen or alloantigens. Some nILs and IL-2s had effects on human monocytes such as inhibiting migration, inducing cytotoxic or growth inhibitory activity against tumor cells, and causing changes in cell surface markers. The IL-2s were also tested for activity in vitro and in vivo in mice. Although there was a 12-fold variation in activity among the preparations, all but one of the IL-2s showed augmentation of the mixed lymphocyte reaction activity and all IL-2s tested stimulated macrophage cytotoxicity in vitro. All IL-2s tested enhanced the mixed lymphocyte-allogeneic tumor cell reaction resulting in greater production of cytotoxic T cells. However, significant quantitative differences in potency were evident among the various IL-2 preparations, especially the nIL-2s. Only very high doses of IL-2 (intraperitoneal injection of 100,000 RU/animal) induced in vivo augmentation of splenic or peritoneal NK cells, although all IL-2s tested increased NK activity against tumor target cells in vitro with substantially lower doses (10-100 RU/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Mononuclear cells from rat spleens that mediate "natural" cytotoxicity are described, and the subpopulation responsible for this activity is partially identified. These naturally cytotoxic cells have been designated "N-cells" and appear to be lymphocytes that lack both detectable immunoglobulin and complement receptors and are therefore not mature B-cells. They are different from the classic "null cells", which have immunoglobulin and/or complement recepters. This subpopulation of cells is responsible for the natural activity present is normal spleens and in spleens from immunized animals. These results indicate that the same subpopulation of cells responsible for natural activity in the short-term assays is also responsible for natural activity in the long-term microcytotoxicity assays reported here. In addition, these mononuclear cells are active against both nonadherent cells and monolayer targets. Natural activity as measured by the long-term assays appears to be somewhat less age specific than that reported previously with the short-term chromium assay. Appropriate base lines are described in an attempt to better document natural activity in these assays. This natural activity must be closely monitored in any system purporting to measure cell-mediated cytotoxicity.

Ten species of purified human leukocyte interferon were tested for their ability to modulate the cytolytic activity of natural killer (NK) cells and the cytolytic and cytostatic activities of monocytes. The interferon species were tested at several antiviral titers and examined for quantitative differences in their ability to modulate immunological function. At the higher doses of interferon (i.e., greater than 500 units) all of the interferon species demonstrated significant augmentation of cytolysis and cytostasis. However, when low levels (i.e., 10-50 units) of interferon were employed, appreciable differences between the various interferon species were seen. A similar pattern of relative potency among the various species of pure leukocyte interferon was seen for augmentation of cytolysis by monocytes and NK cells. In contrast, a different pattern of relative potency was observed for augmentation of cytostasis. These results demonstrated substantial quantitative differences (as much as 100-fold) in the ability of the various species of human leukocyte interferon to induce significant levels of augmentation of these cell-mediated functions. Such results should have significant impact in choosing a specific interferon species for appropriate clinical trials.

Soluble natural killer cytotoxic factors (NKCF) have been detected in the supernatant of normal mouse, rat, and human lymphocytes stimulated in vitro for 1 to 3 days in serum-free medium. Stimulation of large granular lymphocytes (LGL) with NK-sensitive targets or mitogens has resulted in high levels of NKCF production. Previous studies in the mouse and human systems have analyzed the cells responsible for production, specificity, and general characteristics of NKCF. In the present study, using human NKCF as a model for cytolysis by LGL, we have analyzed a variety of agents previously demonstrated to inhibit NK activity. These have included: (i) phosphorylated sugars; (ii) protease inhibitors; (iii) antibodies to rat LGL granules; (iv) Ca++, and Mg++; (v) lipomodulin; (vi) nucleotides; (vii) prostaglandins; and (viii) inhibitors of lysosomal enzymes. All inhibitors were tested for their effects on production of NKCF after target cell interaction, binding of NKCF to target cells, and target cell lysis (after 6-hour NKCF absorption and washing of targets). Phosphorylated sugars and antibodies to rat LGL granules were found to inhibit the lysis of targets by NKCF, whereas the other agents tested had no detectable effect (ATP, cyclic AMP, protease inhibitors, prostaglandin E2). In regard to the production of NKCF, the data indicated that (i) the absence of calcium and magnesium, (ii) prostaglandin E2, and (iii) ATP inhibited production, whereas phosphorylated sugars did not. Studies with these types of agents will now enable us to dissect the sites at which these agents function within the lytic process. In addition to the above studies, purification studies were performed using tritiated arginine to label NKCF to begin biochemical characterization of human NKCF. The results indicated that radiolabeled NKCF has an apparent molecular weight between 20,000 and 40,000. This material demonstrated a pattern of binding to target cells which was similar to the pattern of lysis by NKCF. In addition, the binding of this material was competitively inhibited by unlabeled NKCF preparations. Such approaches with radiolabeled NKCF should be useful for the further study of the biochemical characteristics of human NKCF and of its mechanism of action.

Human effector cells of natural killer (NK) activity have been augmented by pretreatment with a partially purified preparation of interferon (IFN-beta). Using the 4-hr 51Cr release assay to measure cytotoxicity, the boosted effector cells, as well as the spontaneous NK cells, were nonphagocytic cells with receptors for the Fc portion of IgG. Augmentation of cytolytic activity occurred: (1) in medium with either fetal calf serum and human serum; (2) in the presence of absence of monocytes; (3) in both sheep RBC rosette-forming and non-rosette-forming populations; and (4) when only the effector cells were pretreated with interferon. This last observation led to the finding that less than or equal to 5 min of contact at 37 degrees C, 22 degrees C, or 4 degrees C with interferon was necessary for augmentation of cytotoxicity. Augmentation kinetics demonstrated significant boosting of NK activity in peripheral blood leukocytes and highly NK-enriched Percoll fractions after 1 hr of contact with IFN-beta or pure IFN-alpha-Collectively, these results indicate that NK boosting with interferon: (1) occurs after a rapid temperature-independent binding to the effector cells; and (2) has a rapid effect on the cytolytic process.

We have evaluated the methylation state of the T cell receptor beta chain gene (TcR beta) in freshly isolated human large granular lymphocyte (LGL) and T cell DNA in order to investigate the relationship between LGL and T cells with regard to methylation of this region of genomic DNA. In addition, we wished to determine whether hypomethylation of specific regions of the beta chain gene DNA might account for the production of only a nonfunctional 1.0-kilobase (kb) TcR beta mRNA transcript in LGL. Our analysis indicates that the heterogeneous pattern of methylation seen in LGL DNA lies predominantly in the J beta 1 region of the TcR beta DNA structure. The CCGG sequences located at the beginning of the HTF island (CG-rich region) in the J beta 2 region are nonmethylated in both LGL and at least half of the T cell DNA, suggesting that the HTF island is nonmethylated in both LGL and T cell DNA. In addition, specific methylation differences between T cell and LGL DNA can be detected 0.7 kb 3' to the last exon of C beta 1, just 5' to the first exon and within the second exon of the C beta 2 region. The heterogeneous methylation state of the TcR beta J beta 1 region in LGL DNA may be due to and a result of the differential use of the J beta 1 segment for generation of the nonfunctional 1.0-kb mRNA in LGL. These results and our previous studies (Sakamoto, S., Ortaldo, J. R., and Young, H. A. (1988) J. Immunol. 140, 654-660 and Sakamoto, S., and Young, H. A. (1988) Nucleic Acid Res. 16, 2149-2163) indicate that DNA methylation may be one method by which functional gene expression is controlled in specific lymphoid cell populations.

Human large granular lymphocytes (LGL) and T cells were separated from peripheral blood on discontinuous density gradients of Percoll. On a per cell basis, cultured LGL demonstrated higher levels of cytotoxicity against K562 cells than fresh LGL. Cultured T cells acquired cytotoxicity against K562 cells, although they were less cytotoxic than fresh or cultured LGL. During culture, these LGL retained the 3G8, NKH1, OKM1, and OKT10 antigens. Cultured T cells retained the T101 antigen and acquired the OKM1 and OKT10 antigens, but remained negative for the 3G8 or NKH1 antigens. A series of pharmacologic agents known to inhibit cytotoxicity of fresh LGL were also tested for their effects on cultured LGL and T cells. Ethylenediaminetetraacetic acid, known to inhibit the binding of effectors to target cells, inhibited the cytotoxicity of cultured LGL and T cells to the same degree that it inhibited the cytotoxicity of fresh LGL. In contrast, agents which primarily effect post-binding events in cytotoxicity (trypsin, D-mannose 6-P, dexamethasone, prostaglandin E2, anti-LGL granule antibody, and 9.1C3 monoclonal antibody) inhibited the cytotoxicity of cultured LGL and T cells to a much lesser extent than fresh LGL. In addition, on the basis of Michaelis-Menten's kinetic analysis, cultured LGL and T cells developed a higher binding affinity to K562 cells than fresh LGL.

Subpopulations of human peripheral blood mononuclear lymphocytes, after depletion of B-cells and monocytes (by sequential adherence to plastic and nylon wool columns) were separated by Percoll density gradient centrifugation and tested for their ability to proliferate in response to various mitogens, recall antigens, and alloantigens, and to develop cytolytic reactivity in vitro. The low-density fraction [mostly large-granular lymphocytes (LGL)] contained greater than 95% of the total cytotoxic activity of unfractionated nonadherent lymphocytes, against the natural killer (NK)-susceptible K562 target cell, and against other NK-susceptible targets, whereas the high-density lymphocyte fraction (mostly classical T-lymphocytes) demonstrated little or no cytolytic activity against these targets. Conversely, cytotoxic alloreactivity against the lymphoblasts of the donor used for stimulation developed only in the cultures of high density cells. Autologous cytotoxic reactivity, against autologous phytohemagglutinin (PHA)-stimulated lymphoblasts, was not restricted to either subset but developed by both LGL as well as high-density lymphocytes. LGL and high-density T-lymphocytes demonstrated significant proliferative responses to lectins [PHA, concanavalin A (Con A), pokeweed mitogen], and the responses of the LGL were similar in magnitude to those of peripheral blood mononuclear cells or of high-density T-cells. In contrast, only T-lymphocytes responded to the specific recall antigen, purified protein derivative (PPD). These results indicate that LGL are capable of proliferative responses to various lectins, but have no detectable specific memory responses to a soluble antigen. In addition, a different subset of lymphoid cells was responsible for the development of NK-like and specific alloreactivity. Therefore, NK cells and T-cells, although sharing proliferative responses to mitogens, exhibit different function regarding cytotoxic effectors.

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.

Cryopreserved human peripheral blood mononuclear cells (PBMC) were tested for natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) and for high-affinity (29 degrees C) and total (4 degrees C) rosette formation with sheep erythrocytes. PBMC produced variable NK activity following freezing and thawing, but consistently reacted well in ADCC. A significant correlation was found between low NK activity and a decreased percentage of low-affinity rosette-forming cells. On the contrary, the number of large granular lymphocytes (LGL), among which NK cells are restricted, and the reactivity with the monoclonal antibody OKT10, which recognizes the majority of LGL in the peripheral blood, were not significantly altered by cryopreservation. Cryopreserved cells proved to be excellent controls for determining the day-to-day variability of the NK assay and for selecting optimum conditions for this test in the clinical immunology laboratory.

Adoptive cell therapy with various purified populations of human lymphoid and monocytoid effector cells which have been in vitro activated with recombinant interleukin 2 and gamma-interferon was performed in an in vivo nude mouse model of human ovarian cancer. Administration i.p. of human interleukin 2-activated populations of large granular lymphocytes resulted in a significant extension of mean survival time (30 to 60 days) in this ovarian carcinoma model. In addition, T-cells activated with interleukin 2, in a similar fashion to large granular lymphocytes, also significantly prolonged survival of animals with ovarian carcinoma. In contrast, monocytes, with or without gamma-interferon activation, did not improve survival of tumor-bearing mice. In vitro results using direct isotopic release assays to measure efficacy of effectors against the ovarian cancer cells before and after activation, especially the activated natural killer cells, paralleled the effects on survival in the nude mouse model. However, the results with T-cells were somewhat inconsistent in vitro regarding their in vivo efficacy. We assume this is due to a delayed generation of activated killing in T-cells that is generated in vivo. These experimental results in this model system of human ovarian cancer indicate that transfer of autologous activated cells may have a role in the treatment of ovarian cancer patients.

Antigenically different subpopulations of human large granular lymphocytes (LGL) were identified according to their reactivity with monoclonal antibodies (MoAb). Antigen-positive and -negative subsets were isolated by immunoaffinity columns using a Sepharose 4B gel coupled with F(a')2 goat anti-mouse IgG or by flow cytometry cell sorting. The distinct LGL subsets were tested for natural killer (NK) activity against a panel of tumor targets: K562, Daudi, Alab; and for antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated RL male 1 cells. LGL positively selected for any of the following phenotypic markers: B73.1+, OKM1+, OKT11+, and OKT10+ were highly cytotoxic, while B73.1- and OKM1- cells were completely devoid of NK activity. The OKT10- and OKT11- LGL subsets were occasionally cytotoxic, with low levels of reactivity. LGL subpopulations were also tested in a limiting dilution assay (LDA) for their capacity to proliferate in medium supplemented with interleukin 2 (IL-2) and to develop NK-like cytotoxic activity. The majority of proliferative progenitors have the following phenotype: OKT11+, OKM1-, B73.1-, and OKT10-, while the majority of progenitors for cytotoxic cells were OKT11+, OKM1+/-, OKT10+, and B73.1-. Results indicate that although B73.1+ cells can grow, the mature B73.1+ NK cells seem to be primarily derived in vitro from a small subset of less differentiated B73.1 pre-NK progenitors in the peripheral blood lymphocytes.

NK cells are distinct cell populations that can be defined by a series of functional and surface characteristics. The existence of the discrete morphological counterpart for the NK cell, the LGL, has made it particularly convenient for categorizing these effector cells and distinguishing them from a variety of other effector cells. In addition, similar to T cells, considerable diversity exists among the NK cells. The diversity is seen not only in the expression of different surface markers but also within the specificity of target recognition as well as the secretory ability of LGL. It remains possible that much or even all of the heterogeneity that is seen in the phenotype and the function of LGL is due to differences and phases of activation and/or differentiation of these cells. Such differentiation-dependent, non-clonal, heterogeneity appears to account for the heterogeneity seen in subsets of monocytes and macrophages which can be detected in the expression of Ia antigen and Fc gamma receptors on activated macrophages. NK activity is highly regulated by IFNs (alpha, beta) and IL-2. Since NK cells also produce these factors, an autocrine regulation of these cells or their activity may occur. In addition, the mechanism of NK lysis is not yet clearly defined, but seems to involve cytotoxic molecules present in cytoplasmic granules that are released in various forms after effector-target cell contact. In contrast, NC activity against the WEHI-164 target cells appears to be a cytokine-mediated phenomenon, apparently mediated by the release of TNF during the course of the cytotoxicity assay. The mediation of NC activity by the release of TNF may account for the usual requirement for prolonged incubation periods to demonstrate this activity, in contrast to the rapid kinetics of NK activity. The association of TNF with NC activity also provides new clues about the nature of the NC cell, since TNF has been found to be produced predominantly by macrophages but can also be released by lymphocytes.

The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.

The effect of infection with a retrovirus carrying v-raf/v-myc oncogenes (J2 virus) on the in vitro proliferation of human large granular lymphocytes (LGL) was investigated. LGL infected with J2 virus (J2LGL), unlike uninfected cells, grew with a proliferation peak eight days after infection. Such cells retained the morphology and functional properties typical of LGL. Furthermore, 5% of J2LGL produced virus the day after infection, whereas non-virus production was detectable five days later. These data indicate that J2 virus provides a transient mitogenic signal for LGL.

Highly purified human lymphoblastoid interferon derived from virus-stimulated Namalwa cells was administered daily by 6-h i.v. infusion or i.m. injection to 40 patients with a variety of disseminated malignancies refractory to standard therapy. Each patient received escalating doses of 0.1-50 X 10(6) U for up to 5 weeks. Extensive monitoring for changes in various immune functions revealed no sustained elevation in natural killer cell activity, elevated monocyte-mediated antitumor cytostatic activity in approximately 30% of the patients, and depressed lymphoproliferative responses to mitogens and mixed leukocyte culture.

Cells mediating natural killer (NK) activity mediate lysis against a variety of tumor cells and may serve as important effector cells in host resistance to infection. NK active cells are present in virtually all individuals and can be rapidly activated by a wide range of stimuli. Their activities are non-MHC restricted and do not depend on sensitization by antigens, a prerequisite for specific immunity. Several important components of the immunoregulation of NK active cells are: 1) positive or negative signals that regulate the expression of NK activity, and 2) the ability of these cells to function as immunoregulatory cells. The majority of the agents that activate NK activity fall into two basic categories; the interferons and interleukin-2. This review will concentrate on studies regarding these two agents, and will primarily discuss the results obtained with recombinant molecules.

This laboratory has been investigating IFN-gamma gene expression by highly purified human large granular lymphocytes (LGL) and T cells. We report here that within 1 hr after interleukin 2 (IL 2) treatment of freshly isolated human LGL, IFN-gamma mRNA can be detected, with IFN-gamma protein in the culture medium within 4 to 6 hr of treatment. CD3- Leu-11+ LGL require only a single signal for IFN-gamma production because phytohemagglutinin (PHA), phorbol myristate acetate (PMA), IL 2, or ionomycin can each independently induce IFN-gamma production. In addition, PHA and ionomycin (but not IL 2) show significant synergy with PMA as a stimulus to LGL. In contrast, CD3+ T cells require two stimuli for high levels of IFN-gamma production, and not only are PMA plus ionomycin or PHA synergistic, but in addition, IL 2 and PHA demonstrate some synergy. Furthermore, we have found by fractionation of peripheral blood lymphocytes that IL 2-induced IFN-gamma production is associated with the LGL population and not T cells. These results indicate that with certain stimuli, LGL may be the predominant source of IFN-gamma from peripheral blood lymphocytes.

The cytotoxicity inhibition assay is an extremely useful tool for examination of the specificity of cell-mediated cytotoxicity. With this demonstration of cryopreservation of the attacker, target, and inhibitor cells, a powerful new tool is available for the standardization of this assay to better examine the specificity of cytotoxicity assays. Not only can cryopreservation be used to standardize the assay within a single laboratory, but also reagents may now be exchanged between laboratories to better understand the differences in cell-mediated cytotoxicity as observed by different investigators.

Most species of recombinant leukocyte interferons (IFN-alpha A, -alpha B, -alpha C, -alpha D, -alpha F, -alpha I, and -alpha K) were capable of boosting human natural killer (NK) activity after a 2-hr treatment of cells at a concentration of 1-80 units/ml. In contrast, recombinant human IFN-alpha J was found to be incapable of augmenting NK activity after exposure of cells for 2 hr to concentrations as high as 10,000 units/ml. This inability of IFN-alpha J to boost NK activity was not complete because, after exposure of cells to a high concentration of IFN-alpha J (10,000 units/ml) for 18 hr, boosting of cytolysis was observed. IFN-alpha J appeared to interact with receptors for IFN on NK cells since it was found to interfere with the boosting of NK activity by other species of IFN-alpha. In contrast to its deficient ability to augment NK activity, IFN-alpha J has potent antiviral and antiproliferative activities. Such extensive dissociation of these biological activities has not been observed previously with any other natural or recombinant IFN species. Thus, this IFN species may be useful for evaluating the relative importance of various biological activities on the therapeutic effects of IFN, for understanding structure-function relationships, and for determining the biochemical pathways related to the various biological effects of IFN.

Supernatants from the coculture of peripheral blood lymphocytes and the NK-susceptible cell line K562 were highly growth inhibitory for a variety of tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by NK cells. Rather, the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin, a recently described lymphokine. The supernatants of tumor-lymphocyte cultures contained only low levels of IFN-alpha and IFN-gamma, and antibodies to interferons did not affect the observed growth inhibition. The pattern of target cell susceptibility to growth inhibition by this factor was also quite distinct from that seen with purified recombinant LT or TNF. Furthermore, monoclonal antibodies to these cytokines also had no effect on the cytostasis, arguing against a requirement for, or synergistic interaction with, low levels of these cytokines. Some of the targets susceptible to the factor were only growth inhibited but not lysed, thereby distinguishing it from NKCF. Furthermore, the cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which have been reported to block NKCF. Therefore, the results show that a cytostatic factor is released in tumor-lymphocyte incubation that is quite distinct from interferons, LT, and TNF but has characteristics that resemble those of leukoregulin.

We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines. In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and purified large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2. The results indicated that PBL had a 30% increase in cumulative net cell growth and had as high or higher cytotoxic activity after growth in SF medium than in medium containing FBS. Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than in SF medium. However, the cytotoxic activity of cells grown in SF medium persisted during the initial 3 weeks of culture. Purified LGL that were maintained and were subcultured at cell densities of 10(6) cells or greater per milliliter of either SF or FBS-containing medium had equivalent levels of cytotoxicity over a 44-day period in either medium compared with cells subcultured at a density of 5 X 10(5) cells per milliliter of medium. NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxic activity was blocked by 10% FBS. We conclude that the SF medium supplemented with IL-2 can be used as an alternative to FBS-containing medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF.

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.

LGL in addition to mediating natural killer (NK) activity, can secrete a variety of lymphokines, depending on the stimulus used: interleukin-1 (IL-1), interleukin-2 (IL-2), interferon alpha and gamma (IFN), and B-cell growth factor (BCGF). To define more directly whether cells with NK activity can also secrete one or more cytokines, we obtained clones by limiting dilution assays from highly purified preparations of human LGL and cultured them in IL-2-containing medium for several weeks. All the clones tested spontaneously produced detectable levels of IFN-gamma and 35 of 40 clones (87%) produced higher levels when stimulated with PHA. A smaller proportion (16%) of clones (9 of 54) secreted IL-1 after stimulation with LPS, while 34% of the clones (17 of 49) produced IL-2 in response to PHA stimulation. Cytokine production was associated with both cytotoxic and noncytotoxic clones and did not correlate with their surface phenotype, as has been observed for fresh LGL. The ability to produce IL-1 or IL-2 was not usually found within the same clones following PHA and LPS stimulation, respectively; however two clones produced both IL-1 and IL-2 when stimulated in different experiments, but not at the same time. In addition, two of nine cloned LGL simultaneously produced IFN gamma and IL-1. These results indicate that LGL-derived clones have the ability to produce multiple cytokines, suggesting that the LGL population may play an important immunoregulatory role and may also be capable of self-regulation of cytolytic activity.

Acquired immunodeficiency syndrome is associated with a viral (HTLV-III/LAV)-mediated progressive depletion of a helper/inducer T4+ T cell subset, whereas acute T cell leukemia is associated with a viral (HTLV-I)-mediated growth of the same T cell subset. Because large granular lymphocytes (LGL) with natural killer (NK) activity have been shown to spontaneously lyse several virus-infected target cells, the ability of NK cells to lyse both HTLV-I- and HTLV-III/LAV-infected lymphoid cell lines and fresh lymphocytes was explored. Normal lymphocytes (T cells and LGL), with and without pretreatment with recombinant interleukin 2 (IL 2), as well as monocytes, with and without pretreatment with interferon-gamma were employed as effectors. Both IL 2-activated T cells and NK cells were cytolytic for HTLV-I-infected targets. However, only LGL demonstrated significant spontaneous activity against HTLV-I-infected targets. Similarly, LGL showed spontaneous cytolytic activity against HTLV-III/LAV-infected targets, and this cytotoxicity was considerably augmented by IL 2. In contrast, T cells and monocytes were unable to lyse HTLV-III/LAV targets, and only minimal activity was induced by activation. LGL cells, B cells, and monocytes were infectible in vitro by high titers of HTLV-III/LAV. However, levels of reverse transcriptase found in these cultures were significantly lower than the levels in T cell cultures. In contrast, only T cells were susceptible to infection by HTLV-I. Experiments with the use of cell cocultures showed that LGL afforded T cells protection from infection by HTLV-I (as indicated by lack of transformation and viral protein expression) but not from infection by HTLV-III/LAV. Collectively, these results indicate that NK cells may play a role in protecting cells against HTLV infection.

Recombinant human interleukin 2 (rH IL-2) in the presence or absence of additional stimuli, was found to be able to induce and support the proliferation of human peripheral blood lymphocytes (PBLs). These proliferative effects were observed at low doses (less than or equal to 10 U/ml) of interleukin 2 (IL-2) only when additional signals (antigen, mitogen) were provided. However, higher doses (greater than or equal to 100 U/ml) of rH IL-2 significantly stimulated the proliferation of PBL even in the absence of exogenous lectin, antigen, or allogeneic serum. The subpopulation of lymphocytes most responsive to these higher doses of rH IL-2 was the large granular lymphocyte (LGL), the morphologic homologue of natural killer activity. After the separation of human PBLs on discontinuous Percoll gradients, cells from fraction 2 (greater than 90% LGLs) responded in a dose-dependent manner to rH IL-2 alone, whereas cells from fraction 6 (greater than 90% T cells) were only slightly responsive to rH IL-2 alone. A portion of the proliferation of cells from fraction 2 was dependent on the expression of the TAC receptor, because the prior removal of TAC-positive cells significantly reduced IL-2-induced lymphocyte proliferation. These results demonstrate that human LGL that have not been exogenously stimulated can proliferate in direct response to IL-2, and suggest that LGL are the major cellular phenotype in the proliferative response that has been observed clinically.

Human large granular lymphocytes (LGL), which account for virtually all natural killer activity, and T cells were separated from low and high density fractions of Percoll gradients, respectively. We were unable to detect interleukin 2 receptors (IL 2R) on fresh LGL and T cells using flow cytometric analysis with anti-Tac monoclonal antibody or radiolabeled probes with [125I]anti-Tac. IL 2R messenger ribonucleic acid was not observed in fresh LGL or T cells. Although phytohemagglutinin induced the expression of IL 2R on purified LGL and T cells, recombinant IL 2 (rIL 2) alone induced IL 2R messenger ribonucleic acid, IL 2R, and proliferation of LGL but not of T cells. The high level of cytotoxicity of cultured LGL against K562 cells was directly dependent on rIL 2. When T cells were costimulated by phytohemagglutinin and rIL 2 for 3 days, only very low levels of cytotoxicity were generated. Proliferation and cytotoxicity against K562 cells inhibited the culture of LGL in the presence of anti-Tac antibody for 3 days. LGL began to grow more rapidly after 5 days in culture with rIL 2 alone. When rIL 2 were removed from growing LGL for 1 day, proliferation completely stopped, and cytotoxicity was no longer detected. These data indicate that rIL 2 induces IL 2R expression in fresh LGL at the transcriptional level, promoting growth and enhancing cytotoxicity. More importantly, the presence of rIL 2 is necessary and sufficient to induce proliferation of resting LGL and to maintain the growth of LGL with potent lytic activity.

Lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) were administered by the ip route to patients with intra-abdominal malignancies. Pharmacokinetic studies of IL-2 revealed 10- to 100-fold higher concentrations of IL-2 in peritoneal fluid versus serum. Ip levels of IL-2 were maintained well above those required to generate and maintain LAK cells in vitro. LAK cell activity was detectable in the peritoneal fluid for the duration of each treatment cycle and did not disappear until IL-2 was discontinued. Detection of interferon-gamma (IFN-gamma) in the peritoneal fluid of all patients was consistent with production in situ by activated lymphocytes. In some patients, low but detectable levels of IFN-gamma were also found in the serum. In vivo activation of monocytes in the peritoneal fluid as measured by in vitro production of hydrogen peroxide was documented in the majority of patients. Neither interleukin-1 nor tumor necrosis factor-alpha was detected in the peritoneal fluid. We found no correlation between the presence or levels of IL-2, IFN-gamma, or LAK cell lytic activity in peritoneal fluid or serum and response or nonresponse to therapy.

With the use of monoclonal antibodies and continuous flow microfluorometry, we have examined the cell surface antigenic characteristic of rat large granular lymphocytes (LGL) and compared them with monocytes, T cells, and polymorphonuclear leukocytes (PMN). The antigenic profile of rat LGL was of particular interest because these cells have been shown to be highly associated with natural killer (NK) activity. This analysis of enriched populations demonstrated that rat LGL are in antigenically distinct population of cells that share some characteristics with monocytes, T cells, and PMN. Essentially all of the cells in the enriched LGL population expressed with W3/13, OX-8, leukocyte-common (L-C), and asialo GM1 antigens. A portion of these cells were also positive with a monoclonal Ab against the ART-1a antigen. In contrast, few LGL expressed the Ia, W3/25, surface immunoglobulin (sIg), or Thy 1.1 antigens. Monocytes demonstrated a pattern of reactivity similar to LGL but were found to by OX-8 negative. As previously reported, a portion of the T cells were found to express ART-1a, W3/25, and/or OX-8. These results demonstrate that rat LGL are not typical T cells, B cells, monocytes, or PMN. However, the sharing of antigenic markers by LGL with some T cells and monocytes raises the possibility that LGL may be in the development lineage of 1 of these populations.

Utilizing controlled cryopreservation techniques, we were able to standardize the 51Cr release cytotoxicity assay and thereby ensured reliable comparisons between results obtained on different days. Optimal conditions for freezing of both effector and target cells were quite similar. Dimethyl sulfoxide (DMSO) at a concentration of 7.5-10.0% was employed as the cryoprotective agent and cells were frozen at the rate of -1 degrees C/minute. The handling procedures for the cells before and after freezing were important. Factors affecting recovery of functional reactivity were related to toxicity of DMSO for the cells, the osmotic stress placed upon the cells as the DMSO was being removed after thawing, the handling temperature of the freshly thawed cells, and the susceptibility of cells to mechanical damage immediately after thawing. The recovery of lymphocytes after freezing was about 70%; the recovery of cytotoxicity was around 85%. Syngeneic cytotoxic reactivity induced by inoculation with the Moloney strain of murine sarcoma virus was cryopreserved, as were allogeneic cytotoxicity and natural cytotoxic reactivity. Multiple tests employing effector cells from the same frozen pool gave reproducible results; the standard error of the mean percent cytotoxicity was less than 1.5%. Cryopreserved target cells gave decreased day-to-day variability in susceptibility to lysis, since the same population of cells could be employed in each assay. These results demonstrated conclusively that we can now have a constant source of effector cells and target cells, which can be used from assay to assay as an internal standard.

A patient with T-cell chronic lymphocytic leukemia (T-CLL) is reported whose cells demonstrate in vitro suppression of normal lymphocyte mitogen stimulation. The patient, who remains in Rai's clinical stage 0 on no therapy after more than 24 mo of observation, has shown a less aggressive clinical course than is usually attributed to T-CLL. His peripheral blood lymphocytes (PBL) were characterized by functional assays as well as surface markers. Over 90% of the patient's PBL formed rosettes with sheep erythrocytes and were lysed by two T-cell-specific antisera plus complement, while less than 1% bore surface immunoglobulins, and only 3% had complement receptors. In addition, 45% of the PBL demonstrated Ia-like antigens, more than 50% expressed a receptor for the Fc portion of IgG(T gamma), and most of the sheep erythrocyte rosettes were inhibited by theophylline. The patient's cells failed to respond to several mitogens and they caused marked suppression of lymphoproliferative responses to normal PBL to phytohemagglutinin (PHA) and concanavalin A (Con-A). The patient's lymphocytes also exhibited antibody-dependent cytotoxic activity (ADCC) against antibody-coated nucleated target cells, but lacked demonstrable natural killer (NK) activity. This patient's T-CLL cells appear to represent the clonal expansion of a subset of T cells with a previously undescribed pattern of suppressor and cytotoxic activities.

Understanding of the lytic process of NK cells has been greatly advanced in the past decade. However, the definition of the primary event required for lysis has been elusive. The recent enthusiasm about the granule exocytosis/PFP model of membrane damage has been tempered by the multitude of factors that cytotoxic effector cells can secrete and the observation of rapid nuclear degradation before membrane damage which is not well explained by the PFP model. Logically, one would expect that this array of cytotoxic, cytostatic and immunoregulatory factors are secreted for a purpose, and should, therefore, be involved in the lysis of target cells. Further studies are necessary to define the role of each of these factors in plasma membrane and nuclear damage. Additional experiments to determine whether these two events are related will be necessary. On the basis of information currently available, one could hypothesize that after recognition of foreign cells, the NK cell is not single-minded in its delivery of lethal factors. The end result is that target cell lysis may occur because of many factors acting not only on the target cell itself but also on other components of the host immune response.

Recent reports have suggested that the T cell receptor for antigen is somehow involved in the cytotoxicity of natural killer (NK) cells. However, we now report that highly purified, freshly isolated large granular lymphocytes (LGL) from both the human and rat, as well as LGL cultured in the presence of recombinant IL 2, express only the 1.0 kb beta-chain mRNA. The lack of a 1.3 kb mRNA, indicative of a functional beta-chain rearrangement, strongly suggests that a functional T cell receptor beta-chain is absent in freshly isolated LGL, thus making it extremely unlikely that this molecule is involved in target cell recognition by NK cells. These results also suggest that LGL are derived from a lineage distinct from T cells or develop before a functional rearrangement of the T cell receptor beta-chain.

The presence of micromolar concentrations (5 to 100 microM) of adenosine 5'-triphosphate (ATP) in cytotoxicity assays of human natural killer (NK) cells with K562 targets resulted in marked inhibition of NK activity. NK activity of peripheral blood mononuclear cells (PBL) and of purified NK cells (i.e., large granular lymphocytes (LGL] were similarly sensitive to inhibition by ATP. The inhibitory activity was specific to ATP and was not demonstrated with GTP, UTP, CTP, or with other adenosine compounds. This inhibitory activity resulted from an effect of ATP on the effector cells and was not dependent on serum components. ATP did not inhibit binding of the LGL to target cells, and therefore the inhibition by ATP is presumed to be related to some post-recognition metabolic events. Some physiologic role in regulation of NK activity by ATP seems possible, because nonspecific phosphatases (bacterial alkaline phosphatase or human alkaline phosphatase) stimulated NK activity and partially reversed the ATP-induced inhibition of reactivity.

Direct leukocyte migration inhibition assays using the capillary tube technique can be used to demonstrate cell-mediated immunity in vitro. Unfortunately, the cumbersome nature of this technique makes it time consuming and difficult to perform. Similar results have been obtained using the direct agarose microdroplet leukocyte migration inhibition assay. In this paper, modifications of the agarose technique are outlined which insure standardization of droplets and ease of performance of the assay. Additionally a technique is described to reduce the time required for calculation of results.

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.