Blood monocytes and tumor-associated macrophages in human cancer: differences in activation levels.

This study was performed to investigate functional properties of mononuclear phagocytes isolated from ascitic fluid in patients with peritoneal carcinomatosis (PC), and potential immunomodulatory effects of soluble factors produced or induced by human metastatic malignant cells. Phagocytic activity and nitric oxide production of peripheral blood monocytes (PBMo) and tumor-associated macrophages (TAM) or peritoneal macrophages (PEM) were synchronously examined in cancer patients and control individuals. Our results showed that contrary to peripheral blood monocytes, where phagocytic activity was not altered, TAM had impaired phagocytic activity. Moreover, dilutions of crude supernatant from short-term cultures of the peritoneal cells obtained from ascitic fluid of patient with PC, cause a significant, dose dependent inhibition of control PBMo and PEM phagocytosis, comparable to those in TAM, indicating that a soluble factor(s) plays a prominent role in this alteration. Next, we investigated the potential of cancer patients mononuclear phagocytes to produce nitric oxide (NO). It was found that TAM produce fourfold lower levels of NO than PEM from control subject, whereas monocytes produce NO at levels comparable to those of corresponding controls. These data support the hypothesis that depressed TAM function may contribute to the mechanisms of tumor escape from immune destruction.

Measurement of NK activity by the microcytotoxicity assay (MCA): a new application for an old assay.

Natural killer (NK) cells are spontaneously cytotoxic immune effector cells with the ability to selectively destroy tumor cells without harming normal cells. To perform this function, NK cells utilize two main cytotoxicity pathways, the well known perforin/granzyme-mediated secretory/necrotic killing and the recently defined TNF family ligand-mediated non-secretory/apoptotic killing. The former mechanism is manifested mainly against a few cultured leukemia cell targets, while the latter mediates killing against a large variety of tumor cell targets. Therefore, the biological role and significance of these mechanisms might be different. The NK cell-mediated necrotic killing has been reliably and selectively measured in humans by the standard 4-h 51Cr release assay (CRA) against K562 myeloid leukemia cell targets. However, no standardized high throughput assay is available for testing the NK cell-mediated apoptotic killing. Here, we introduce the modified MCA as a convenient method for measuring perforin/granzyme-independent NK cell-mediated apoptotic killing. The assay is performed in microwells of Terasaki tissue culture microtest plates, using adherent tumor cell targets, which are selectively susceptible to non-secretory/apoptotic killing and resistant to secretory/necrotic killing mediated by NK cells. Target cells are plated in microwells and incubated overnight to adhere to the plastic surface and to regenerate cell surface-bound TNF family receptors. Following this adherence, target cells are co-incubated with freshly isolated human peripheral blood mononuclear leukocytes (PBMNL) or purified subpopulations of immune cells for 24 h in various effector/target (E/T) ratios. During this incubation, dead target cells become non-adherent and are removed by washing the wells. Remaining adherent (viable) target cells are fixed, stained and optically counted. A notable dose-dependent (peak at 200:1 E/T ratio), time-dependent (peak at 24 h of incubation) and donor-dependent killing of tumor cells was consistently and reproducibly induced by PBMNL of normal donors. Using purified subpopulations of immune cells, it was demonstrated that among PBMNL, CD3(-)CD56(+)CD16(+) mature NK cells are the only mediators of tumor cell killing in MCA, as well as in CRA. Comparative studies of NK activity detected by MCA and CRA, performed with PBMNL from normal individuals and breast cancer patients, showed no significant correlation between the cytotoxicities measured in the two assays. In addition, while NK activity measured in CRA was normal in most breast cancer patients, NK activity assessed in MCA was decreased in a large majority of the patients. Thus, MCA is a sensitive NK assay, which is biologically different from CRA, and may be clinically relevant. MCA has also a higher throughput, and is more practical and economical than CRA.

Role of TNF family ligands in antitumor activity of natural killer cells.

NK cells have the ability to destroy tumor cells by two main cytotoxic pathways, the well-known perforin/granzyme-mediated secretory/necrotic killing and the newly defined TNF family ligand-mediated apoptotic killing. The former mechanism is operative mainly against a few cultured leukemia cell targets, while the latter mediates substantial activity against most tumor cell targets. It also appears from emerging data that the apoptotic mechanism is the main antitumor pathway in vito. This review is focused on the apoptotic mechanism of killing, the molecules and cell signaling pathways involved in this process, and its potential biologic significance along with its relation to the secretory/necrotic cytolytic pathway.

Inhibition of apoptosis in human tumour cells by the tumour-associated serpin, SCC antigen-1.

The squamous cell carcinoma antigen (SCC Ag) is a tumour-associated protein and a member of the serine protease inhibitor (serpin) family. The SCC Ag has been used as a serologic tumour marker for SCC progression, and its elevated serum levels are a risk factor for disease relapse. However, the biologic significance of this intracytoplasmic protein in cancer cells remains unknown. In this report, we demonstrated that apoptosis induced by 7-ethyl-10-hydroxycamptothecin, tumour necrosis factor-alpha (TNF-alpha) or interleukin (IL)-2-activated natural killer (NK) cells was significantly inhibited in tumour cells transduced with the SCC Ag-1 cDNA, as compared to control cells in vitro. Also, inhibition of the SCC Ag-1 expression in tumour cells by transfection of antisense SCC Ag-1 cDNA was accompanied by significantly increased sensitivity of these cells to apoptosis induced by etoposide or TNF-alpha. The mechanism of protection of tumour cells from apoptosis involved inhibition of caspase-3 activity and/or upstream proteases. In vivo, tumour cells overexpressing the SCC Ag-1 formed significantly larger tumours in nude mice than the SCC Ag-1-negative controls. Thus, overexpression of the SCC Ag-1, a member of the serpin family, in human cancer cells contributed to their survival by mediating protection from drug-, cytokine- or effector cell-induced apoptosis.

Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells.

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-alpha, LT-beta, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-alpha1beta2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-alpha1beta2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.

Rat NKR-P1+ CD3+ T cells: selective proliferation in interleukin-2, diverse T-cell-receptor-Vbeta repertoire and polarized interferon-gamma expression.

Cells expressing markers of both natural killer and T lymphocytes (NK T cells) in humans and mice express a restricted T-cell receptor (TCR) repertoire, are of CD4- CD8- or CD4+ CD8- phenotype, and upon anti-CD3 stimulation secrete large amounts of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). NK T cells may be the primary source of IL-4-promoting T helper type 2 (Th2) responses and/or they might be involved in regulating the balance between Th1- and Th2-type immune responses, and may consequently affect susceptibility to autoimmune diseases associated with a skewed Th phenotype. We show that rat NK T cells selectively proliferate to IL-2, and use this fact to analyse cytokine production by NK T cells in two rat strains differentially susceptible to Th1- or Th2-type autoimmune diseases. Analysis by reverse transcription-polymerase chain reaction revealed that, in contrast to mouse, rat NK T cells secrete exclusively IFN-gamma and not IL-4 after anti-CD3 stimulation, and use a wider TCR-Vbeta repertoire, suggesting that rat NK T cells are not essential for the development of Th2-type CD4+ T-cell responses.

Expansion of functional NK cells in multiple tissue compartments of mice treated with Flt3-ligand: implications for anti-cancer and anti-viral therapy.

The generation and activity of NK cells appear to be regulated by a particular set of cytokines. We examined the in vivo effects of recombinant human Flt3 ligand (Flt3-L), a recently cloned potent hemopoietic cytokine, on NK cell development in mice. Daily i.p. administration of Flt3-L consistently induced striking increases in both the absolute number and the total cytotoxic activity of mature nonactivated NK cells within various tissues. Dose- and time-dependent increases were observed in the bone marrow (approximately 2- and approximately 11-fold, respectively), thymus (approximately 2.8- and approximately 2.0-fold), blood (approximately 11- and approximately 15-fold), spleen (approximately 10- and approximately 9-fold), and liver (approximately 15- and approximately 39-fold). In addition, IL-2 induced a rapid increase in NK activity, NK cell proliferative responses, generation of lymphokine-activated killer activity, and development of activated adherent NK cells, which were all significantly increased by Flt3-L treatment. Thus, in addition to its recently reported capacity to stimulate dendritic cell production, Flt3-L has a prominent biologic role in NK cell generation in vivo. This is probably a result of selectively induced expansion of NK cell progenitors (pro-NK cells), because Flt3-L stimulates in vitro proliferation of pro-NK cells without affecting the cytotoxicity of mature NK cells. The results also indicate that either alone or in combination with a potent activator of NK cells, such as IL-2, Flt3-L could be used to markedly augment the number and activity of NK cells, especially in the liver. Flt3-L appears to have considerable potential for therapy of both cancer and viral infection.

Human tumor antigen-specific T lymphocytes and interleukin-2-activated natural killer cells: comparisons of antitumor effects in vitro and in vivo.

Human antitumor effector cells include class I major histocompatibility complex (MHC)-restricted T cells and non-MHC-restricted natural killer (NK) cells. These two types of effector cells have not been directly compared for the ability to eliminate tumor cell targets. Here, we compare in vitro and in vivo antitumor functions of two human T-cell lines specific for a shared tumor antigen to the antitumor functions of A-NK cells, a subset of IL-2-activated NK cells. Human squamous cell carcinoma of the head and neck cell lines cultured in suspensions or as spheroids or tumor xenografts established in nude mice were used to evaluate antitumor functions of IL-2-activated and expanded T and NK effector cells in various assays, both in vitro and in vivo. Both tumor cell targets, PCI-13 and OSC-19, expressed class I and II MHC antigens after IFN-gamma pretreatment, gave rise to tumors upon injection into immunosuppressed nude mice, and were resistant to lysis by resting NK cells but sensitive to lysis mediated by A-NK cells or HLA-A2-restricted T-cell lines specific for a shared squamous cell carcinoma of the head and neck antigen. No significant differences were observed in the ability of A-NK cells or tumor-specific T cells to bind to tumor cell monolayers or to enter into spheroids. However, A-NK cells mediated significantly higher killing than tumor-specific CD8+ T cells in 4-h 51Cr-release assays (a measure of cell membrane damage and necrosis), 1-h [3H]thymidine-release assays (a measure of DNA fragmentation and apoptosis), and in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays (a measure of apoptosis). In contrast, CD8+ T cells were consistently more effective than A-NK cells in inducing growth inhibition of tumor cells in 24-h MTT assays. In the presence of tumor-specific antibodies, A-NK cell binding, entry into spheroids, and infiltration into tumor in vivo were significantly increased. In vivo perilesional delivery of effector cells to mice with established tumors indicated that human A-NK cells exert antitumor effects as potent as those of tumor-specific T cells. However, in contrast to tumor-specific T cells, A-NK cells are readily available for cancer therapy, expand rapidly in culture without prior sensitization, and can be armed with antitumor antibodies to increase localization of effector cells to the tumor.

Natural killer cells and tumor therapy.

Evidence has been reviewed which indicates that NK cells play a role in the control of metastasis dissemination. Both activation of endogenous NK cells in a tumor-bearing host and adoptive transfer of ex vivo activated NK cells may be therapeutically beneficial. The small number of phase I/II clinical trials of AIT with A-NK cells performed in patients with cancer so far does not allow firm conclusions, except to ascertain the feasibility and a lack of toxicity of this form of therapy. Although numerous trials have been performed with BRMs, many of which are known to upregulate NK activity in vivo, a general lack of correlations between clinical responses or survival and upregulated NK activity in the peripheral blood has dampened enthusiasm for biological therapies. However, these clinical trials have been confined largely to patients with advanced metastatic disease. It is highly likely that tumor-induced immunosuppression plays a crucial role in neutralizing the benefits of BRM therapy, and that levels of effector cell activation sufficient for metastasis elimination are seldom achieved in this clinical setting. On the other hand, administration of BRMs in the adjuvant setting could be more effective and when combined with monitoring for effector cell functions might perhaps provide a better guide for achieving the levels of endogenous NK activity necessary for elimination of remaining or occult metastases. An improved understanding of NK cell biology in cancer patients is likely to serve as a positive reinforcement for design of a new generation of clinical trials incorporating novel approaches to NK cell mediated cancer therapy.

Absence of B7.1-CD28/CTLA-4-mediated co-stimulation in human NK cells.

Recent studies have suggested that B7-CD28 interactions provide co-stimulatory signals for activation of NK cells. Transduction of the B7.1 (CD80) gene into tumor cells has been shown to trigger proliferation and cytotoxicity of murine NK cells and a human NK cell line, YT2C2. Therefore, transduction of the B7.1 gene into CD80-negative human squamous cell carcinomas of the head and neck (SCCHN) and its stable expression was expected to upregulate proliferation and cytotoxic activities of human NK cells. However, expression of the B7.1 receptors, CD28 and CTLA-4, could not be demonstrated on the surface or in the cytoplasm of normal human NK cells, irrespective of the state of their activation. In proliferation experiments or various cytotoxicity assays, utilizing highly purified human NK cells as responder or effector cells, no enhancement of NK cell generation or activity, respectively, by B7.1+ SCCHN was observed relative to non-transduced or LacZ gene-transduced SCCHN. In contrast, co-incubation of B7.1+ SCCHN targets with human NK cells induced significant inhibition of NK cell growth. Thus, the B7.1-CD28/CTLA-4 pathway is not involved in triggering of human adult NK cells.

The in vivo effect of hepatotrophic factors augmenter of liver regeneration, hepatocyte growth factor, and insulin-like growth factor-II on liver natural killer cell functions.

Fine balanced sequential changes of the levels of circulating hepatotrophic factors are essential for normal liver regeneration. Our recent studies have indicated that liver-resident natural killer (NK) cells are important regulators of liver regeneration and have raised the possibility that hepatotrophic factors might mediate their activities through NK cells. In the present study, we assessed the effects of in vivo administration of three hepatotrophic factors (augmenter of liver regeneration [ALR], insulin-like growth factor-II [IGF-II], and hepatocyte growth factor [HGF]) on NK cells in normal rats. Each of the three, given over a 1-day period in doses known to produce hepatotrophic activity, induced inhibition of NK cell cytotoxic activities in the population of mononuclear leukocytes (MNL) in the liver, but not in MNL from the spleen or peripheral blood. In contrast to these results obtained by the whole animal treatment, the three molecules had no effect on NK cell functions when added to cultures of MNL from the livers, spleens, or blood of untreated rats. These data support and extend our previously advanced hypothesis that ALR and other hepatotrophic factors play an important role in liver regeneration by regional regulation of NK cells through some as-yet-unknown intermediary mechanism.

Nonsecretory apoptotic killing by human NK cells.

We defined a novel constitutive mechanism of cell-mediated cytotoxicity, utilized by nonactivated human peripheral blood NK cells against a variety of tumor cell targets resistant to secretory (i.e., perforin/granzyme-mediated) NK cell killing. Untreated NK cells rapidly induced membrane damage and necrosis in K562 target cells (as determined by 51Cr release assay and confirmed by transmission electron microscopy; TEM), in the absence of DNA fragmentation and apoptosis (as assessed by [3H]thymidine release assay and TEM). Chelation of extracellular Ca2+ or paraformaldehyde fixation completely abrogated NK cell secretory activity and necrotic killing. In contrast, NK cells with either normal or impaired secretion consistently exhibited cytotoxicity against a wide variety of solid tumor cell lines in 1-h [3H]thymidine release, but not in 4-h 51Cr release, assays. Thus, the cytotoxicity was attributable to a nonsecretory, cell membrane-mediated mechanism. It appeared to selectively induce DNA fragmentation and apoptosis without plasma membrane damage and necrosis. This was further demonstrated by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and confirmed by TEM. Among defined populations of immune cells, only NK cells showed an inherent ability to rapidly induce apoptotic death in solid tissue-derived malignant cells. This study demonstrates that NK cells are unique immune cells constitutively endowed with multiple mechanisms of destroying abnormal cells.

Elimination of established liver metastases by human interleukin 2-activated natural killer cells after locoregional or systemic adoptive transfer.

An in vivo model of liver metastasis induced by human gastric carcinoma was established in nude mice and used for locoregional or systemic immunotherapy with a subset of human A-natural killer (NK) cells defined previously. A single intrasplenic (i.s.) delivery of A-NK cells (1 x 10(7)) and interleukin 2 (IL-2; 60,000 international units, twice a day for 5 days, i.p.) to animals with 3-day established liver metastases, but not IL-2 alone, resulted in rapid (within 24 h) elimination of the majority of metastases and significantly improved survival. A single i.s. or i.v. transfer of these effector cells and IL-2 significantly prolonged survival of the mice with 3-day established metastases (P < 0.03 and P < 0.02, respectively) compared with untreated mice. Using 51Cr-labeled A-NK cells, it was determined that, at best, 75% of 1 x 10(7) cells delivered i.s., and up to 50% of those delivered i.v. were found in the liver 30 min-4 h later. Using image analysis with Di-O dye-labeled A-NK cells, 60-100% of A-NK cells delivered i.s. or i.v. were detected in the liver 24 h later. By light microscopy, 3-day liver metastases were mostly intravascular, but some had already begun to spread into liver tissue. When rhodamine- or Di-O dye-labeled A-NK cells were injected i.s. or i.v. to study their distribution in the liver, they were detectable by confocal fluorescence microscopy in tumor-free tissue and in association with tumor cells 12-24 h after transfer. No evidence for selective localization of A-NK cells to liver metastases was obtained; many A-NK cells were randomly distributed in tissue and not associated with visible metastases. However, confocal fluorescence and electron microscopy showed some A-NK cells to be in cell-to-cell contact with tumor cells, both in the blood vessel and liver tissue. These results indicate that a majority of human A-NK cells transferred i.s. or i.v. to mice with liver metastases have the capacity to migrate to the liver and to enter liver tissue and tumor metastases in vivo. The presence of these effector cells even in a modest number in the liver leads to elimination of most, but not all, metastases and to significantly prolonged survival of animals treated with A-NK cells and IL-2.

Changes of liver-resident NK cells during liver regeneration in rats.

To determine the role of NK cells in regulation of tissue growth, the phenotype and function of liver-resident NK cells were studied after 70% partial hepatectomy in rats. The process of liver regeneration was generally completed by day 14. In contrast, the number of liver-resident NK cells (NKR-P1bright) was restored as early as day 3 after partial hepatectomy. However, spontaneous functions of liver-resident NK cells, including killing of YAC-1 and P815 targets, Ab-dependent cellular cytotoxicity, and redirected killing via NKR-P1, were continuously suppressed throughout the entire period of liver regeneration (from 3 h to 14 days). Augmentation of NK cytotoxicity against P815 targets and induction of NK cell adherence to plastic following 24 h of IL-2 stimulation showed a similar pattern of suppression. However, IL-2-induced augmentation of YAC-1 killing, proliferation and generation of adherent NK cells, and LAK activity in 5- to 7-day cultures were found to be suppressed only during the first 24 h and increased between days 2 and 7 after hepatectomy. Sorted NK cells (> or = NKR-P1bright) from liver-resident mononuclear leukocytes 24 h after partial hepatectomy showed the same pattern of suppression as unsorted mononuclear leukocytes. In contrast to liver-resident NK cells, no significant changes were detected in peripheral blood or spleen NK cells of rats following partial hepatectomy. Of particular interest, in normal liver, hepatocytes were resistant to NK lysis, while resident NK cells were cytotoxic for various NK-sensitive targets. In contrast, during the early period of liver regeneration, when hepatocytes were sensitive to lysis by liver-resident NK cells of normal rats, NK cells obtained from regenerating liver tissues were unable to mediate cytotoxicity. At the final phase of liver regeneration (days 7-14 after hepatectomy), both resistance of hepatocytes to killing by NK cells and cytotoxicity of liver-resident lymphocytes against hepatocytes from regenerating liver were simultaneously restored. In vivo depletion of NK cells by injection of rats with anti-NKR-P1 mAb resulted in a significant augmentation of liver regeneration subsequent to partial hepatectomy. Our data suggest that liver-resident NK cells may be involved in regulation of the extent of liver regeneration.

Antitumor activities of subsets of human IL-2-activated natural killer cells in solid tissues.

Human NK cells can be separated into two functionally distinct subpopulations based on the ability to rapidly respond to IL-2 by adherence to solid surfaces. To determine functions of the NK cell subsets in solid tumor tissues, adherent (A) and nonadherent (NA) NK cells were evaluated for their ability to infiltrate multicellular tumor spheroids in vitro, to kill carcinoma (CA) cell targets in these spheroids, and to mediate antitumor activity in vivo. A-NK cells were less cytolytic than NA-NK cells against CA targets in single cell suspensions or in monolayers. However, A-NK cells showed a significantly better ability than NA-NK cells to infiltrate tumor tissues and kill tumor cells in spheroids of human squamous cell CA of the head and neck or breast CA. Perilesional delivery of human A-NK cells and IL-2 resulted in regression of established human squamous cell carcinoma of the head and neck tumors growing subcutaneously in immunosuppressed nude mice. Similarly, in a xenograft model of human gastric CA metastatic to liver of nude mice, a single intrasplenic injection of A-NK cells in combination with i.p. infusions of IL-2 significantly reduced the number of established hepatic metastases (p < 0.007) and prolonged survival of the mice (p < 0.003). In contrast, NA-NK cells were ineffective in either of the in vivo xenograft tumor models. These findings demonstrate that A-NK cells represent a biologically unique and important subset of NK cells that, in contrast to the rest of NK cells, function as effector cells in solid tumor tissues and, consequently, have a great antitumor therapeutic potential.

Immunotherapy of liver metastases of human gastric carcinoma with interleukin 2-activated natural killer cells.

To better understand the events occurring during immunotherapy of liver metastases with effector cells, we have developed a clinically relevant animal model in which both effector-tumor cell interactions and survival can be evaluated. A cell line of human gastric carcinoma (HR) metastatic to the liver has been established from a patient's liver biopsy. HR cells (10 x 10(6)) injected intrasplenically metastasize into the liver of immunosuppressed nude mice, with micrometastases detectable histologically by day 4 and macrometastases by day 7. The animals subsequently develop ascites and die between days 30 and 40 after tumor injection. To investigate early metastatic events in the liver, HR cells were transduced with a plasmid containing both the lacZ gene under the control of the CMV promoter and NeoR gene. Transfectants selected for neomycin resistance were lacZ gene positive and stained blue in the presence of a beta-galactosidase substrate, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). These transfectants (HRLZ) remained lacZ gene positive for at least 25 passages in vitro. Injected intrasplenically, an HRLZ clone grew invasively in nude mice and formed liver metastases comparably to parental tumor cells. The number and localization of blue X-Gal-positive tumor cells were followed in liver tissues of animals sacrificed at various times, from 1 h to 28 days postinjection of HRLZ cells. HRLZ cells were seen in liver blood vessels and sinusoids within 1 h after injection, and the progressive growth of micrometastases and macrometastases could be followed with precision by X-Gal staining. On day 3 after injection of HRLZ cells, numerous micrometastases were established containing 12-16 tumor cells. When these 3-day established HRLZ micrometastases were treated by the intrasplenic infusion of interleukin 2 (IL2)-activated human natural killer (NK) cells selected by IL2-induced adherence to plastic (A-NK) and systemic IL2, nearly all liver micrometastases were eliminated within 24 h after a single transfer of A-NK cells (P < 0.001). This xenogeneic model was also used for adoptive immunotherapy of 7-day established liver macrometastases with human A-NK cells injected intrasplenically and exogenous IL2 given i.p. A significant decrease in the number of hepatic metastases and the weight of livers (P < 0.003) in comparison with those of control mice was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Distinct phenotypic and functional characteristics of human natural killer cells obtained by rapid interleukin 2-induced adherence to plastic.

Human natural killer (NK) cells can be functionally subdivided into adherent (A) and non-adherent (NA) subpopulations. In the presence of 22 nM of interleukin 2(IL2), a substantial proportion of resting (R)-NK cells developed adherence to plastic as early as after 5 min of IL2 incubation, and by 1-5 hr of IL2 induction, 16% (range, 4-30%) of NK cells were adherent. Optimal concentration of IL2 for adherence of NK cells was 2-22 nM. This adherence was blocked completely by antibody to IL2 receptor (IL2R)-beta and, partially, by antibodies to beta 1 or beta 2 integrins, ICAM-1, CD2 or LFA3, but not by antibodies to the IL2R-alpha or CD56 antigen. A-NK cells separated from NA-NK cells after 5 hr of incubation in the presence of IL2 were significantly (P < 0.05) enriched in CD56dimCD16dim or -IL2Rp55+ and IL2Rp75+ cells, but were depleted of CD56bright CD16- cells. While surface density of CD56 and CD16 antigens was lower, that of beta 2 integrins (CD18, CD11a, CD11b) was higher on A-NK than on NA-NK cells. In a single-cell cytotoxicity assay, 61% of A-NK vs 37% of NA-NK cells bound, and 24% of A-NK vs 11% of NA-NK cells killed, K562 targets. In 4-day cultures with 0.02 or 2.2 nM of IL2, A-NK cells developed lymphokine-activated killer (LAK) activity later than NA-NK cells. By autoradiography, three to eight times more A-NK than NA-NK cells incorporated [3H]TdR into cell nuclei between 48 and 96 hr of IL2 incubation. In 14-day cultures in the presence of 22 nM of IL2, A-NK cells, which were initially adherent but later grew as single-cell suspensions, proliferated better (30-fold; P < 0.03) and expressed lower membrane density of CD56 than NA-NK cells. In culture, A-NK cells had consistently higher cytotoxicity against K562 targets than NA-NK cells, but cytotoxicity against Daudi was similar for both subsets. The data indicate that short incubation (1-5 hr) of human NK cells in the presence of 22 nM of IL2 allows for selection of a subpopulation which differs from the rest of NK cells not only by properties of rapid adherence to plastic, but also by a characteristic phenotype (CD3-CD56dim or -CD16dim or -beta 2integrinsbrightIL2Rp75+), rapid expression of IL2R-alpha, higher NK activity, delayed development of LAK activity, and ability to respond optimally in the presence of 22 nM of IL2.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid Il-2-induced adherence of human natural killer cells. Expression of mRNA for cytokines and IL-2 receptors in adherent NK cells.

Natural killer (NK) cells selected by IL-2-induced rapid adherence to plastic and called A-NK cells represent a phenotypically and functionally distinct subset of mature peripheral blood NK cells. To further characterize this subset of NK cells functionally, their potential to express mRNA for the IL-2R and various cytokines after IL-2 activation was examined. Highly purified normal human peripheral blood resting NK (R-NK) cells were obtained by negative immunoselection using OKT3 mAb and magnetic beads coated with goat anti-mouse Ig. By two-color flow cytometry, > 90% of these R-NK cells were either CD3-CD56+CD16+ or - or CD3-CD56-CD16+. R-NK cells were activated in the presence of 6000 IU/ml (22 nM) of IL-2 for different periods of time. After 1, 3, 5, or 24 h, plastic-adherent (A) and nonadherent (NA) NK cells were separated and compared for the expression of the IL-2R or cytokine mRNA by in situ hybridization, using 35[S]-cDNA probes. Only low proportions of R-NK cells expressed genes for IL-2Rp55 (16%) or cytokines IL-2 (20%), IFN-gamma (18%), TNF-alpha (16%), and TGF-beta (7%). Thus, the genes for the IL-2Rp55 and these cytokines were not constitutively expressed by most human R-NK cells, and there was no indication that the NK cells used in these experiments were activated in vivo or during the purification procedure. However, larger proportions of R-NK cells showed expression of mRNA for IL-1-beta (35%) and IL-6 (40%), which indicates that genes for these cytokines may be constitutively expressed in a substantial proportion of normal human circulating NK cells. When R-NK cells were incubated in the presence of 22 nM of IL-2 for 1 to 24 h and separated into A-NK cells and NA-NK cells, a large proportion of A-NK cells became positive for IL-2R and cytokine gene expression. In contrast, the proportion of mRNA-positive NA-NK cells was similar or lower than that observed for R-NK cells, with the exception of an increase in TGF-beta.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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