Syngeneic pregnancy induces overexpression of natural killer T cells in maternal liver

Conditions such as stress, infection, autoimmune disease, etc. elevate the number and function of extrathymic T cells that are generated mainly in the liver. As primitive, self-reactive clones of T cells that coexpress receptors of the natural killer (NK) lineage, they mediate cytotoxicity against altered self, malignant and infected cells and have the unique potential to rapidly secrete large amount of T helper 1 (Th1) or Th2 cytokines. To elucidate whether some of these changes occur even during the syngeneic pregnancy, we made phenotypic and functional characterization of mononuclear lymphatic cells (MNLCs) isolated from the liver and spleen of pregnant C57BL/6 mice, testing their cytotoxicity against syngeneic thymocytes as well as against NK- and lymphokine-activated killer (LAK)-sensitive targets. The data have shown that on the sixteenth day of syngeneic pregnancy TCRint, NK1.1+ and IL-2Rbeta+ cells were accumulated in the liver, while the quantities of CD4+ and CD8+ T cells and total number classical NK (NK1.1+CD3- or IL-2Rbeta+CD3-) cells were increased in the spleen. Pregnancy-activated hepatic and splenic MNLCs were more cytotoxic against syngeneic thymocytes, YAC-1 and P815 targets, suggesting that the maternal liver is a main producer of autoreactive NKT clones, which subsequently augment NK- and LAK cell-mediated cytotoxicity in the liver and spleen.     

Early appearance and activation of natural killer cells in tumor-infiltrating lymphoid cells during tumor development

We investigated NK cell infiltration into tumor developing lesions at early stage of tumor development after intraperitoneal inoculation of 3LL lung carcinoma into syngeneic C57BL/6 mice. Natural killer (NK) cells, which were detected by anti-NK 1.1 monoclonal antibody (mAb), remarkably increased in number in tumor-developing lesions (peritoneal cavity) as early as day 3 after inoculation of 3LL. The tumor-infiltrating NK cells from 3LL-inoculated mice produced a high level of interferon-γ by co-culture with 3LL and showed enhanced cytotoxic activities against both NK-sensitive (YAC-1) and NK-resistant (3LL and P815) tumors. Furthermore, mice depleted of NK cells by injection of anti-NK 1.1 mAb or antiasialo GM1 antibody showed shorter survival times after intraperitoneal inoculation of 3LL when compared with control mice. These results suggest that NK cells infiltrate the tumor-developing lesion at an early stage and may participate in the early protection against tumors through production of a high amount of interferon-γ and enhanced cytotoxicity at tumor-bearing sites.     

Bone marrow transplantation in the rat. Lytic functions of inflammatory leukocytes during acute graft-versus-host disease

We have isolated inflammatory leukocytes from various lymphoid and parenchymal organs after total body irradiation and bone marrow transplantation from either an allogeneic or syngeneic strain and tested their ability to perform lytic functions in vitro. No direct lytic activity (i.e. cytotoxic T lymphocytes, CTL) to relevant strain-derived target cells in the lymphoid or parenchymal target organs was seen preceding or during acute graft-versus-host disease (aGVHD). Instead, the leukocytes of the spleen and blood and the inflammatory cells of liver and lungs were efficient effector cells against recipient-derived target cells in the presence of relevant antibody (antibody dependent cellular cytotoxicity, ADCC). The NK activity against YAC-1 (natural killer, NK) target cells was first high in the spleen, but when the aGHVD appeared in the allograft marrow recipients the NK activity decreased in the spleen with a concomitant increase in the liver, but not in the other parenchymal target organs. At the same time no NK activity was seen in the syngeneic marrow graft recipients' parenchymal organs. These observations suggest functional differences in the structure of inflammation in the different target organs of aGVHD.     

Syngeneic B lymphoma cells provide a unique stimulus to natural killer (NK) cells in genetically low-NK SJL/J mice

SJL/J mice are a genetically low-NK strain, and their cytotoxic activity cannot be augmented with conventional NK inducers. In contrast, effector cells taken from the lymphoid tissues of SJL mice bearing a syngeneic B cell lymphoma (RCS) show variable, but significant levels of cytotoxic activity against NK-susceptible targets, such as YAC-1. Previous results suggested that the RCS cells themselves contributed to this cytotoxicity. However, results presented here indicate the most, if not all of the activity present within the lymphoid tissues of RCS-bearing mice is mediated by RCS-activated, host NK cells. These results were confirmed by in vitro studies, which demonstrate that both gamma irradiated (gamma-) RCS cells and gamma-allogeneic spleen cells induce cytotoxic activity in SJL spleen cells against YAC targets. However, the cytotoxicity induced by gamma-allogeneic cells is mediated largely by lymphokine-activated killer (LAK) cells, since these effectors also lyse NK-resistant target cells, such as L1210. In contrast, the cytotoxic effector cells that are induced by syngeneic gamma-RCS cells cause lysis of YAC targets, but not L1210 target cells. These data indicate that the syngeneic B cell lymphomas of SJL mice are a unique stimulus for host NK cells in this strain. Since activated NK cells produce a variety of lymphokines, RCS stimulation of host NK cells in SJL mice may provide some of the growth-promoting lymphokines that are known to be necessary for progressive growth of these lymphoma cells.     

The differential effect of stress on natural killer T (NKT) and NK cell function

When C57Bl/6 mice were exposed to restraint stress for 12 h or 24 h, lymphocytopenia was induced in the liver, spleen, and thymus. We examined which types of lymphocytes were sensitive or resistant to such stress by a immunofluorescence test. T cells of thymic origin were sensitive while NKT and NK cells were resistant. In contrast to the increase in the proportion of NK cells, NK activity of liver lymphocytes against YAC-1 targets decreased at 24 h after stress. On the other hand, their NKT cytotoxicity against syngeneic thymocytes increased in parallel with an increase in their proportion. In perforin -/- B6 mice and B6-gld/gld (Fas ligand-) mice, NK cells were found to mediate cytotoxicity through perforin while NKT cells mediated self-reactive cytotoxicity through Fas ligand. These results suggest that stress increases the proportion of both NK and NKT cells, but that NK cytotoxicity is suppressed while self-reactive NKT cytotoxicity is not, due to a diversity of their functional mechanisms.     

Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity

Interleukin 12 (IL-12)-activated NK1.1+TCRalpha beta+ (NKT2) and NK1.1+TCRalpha beta- (NK) cells exhibit cytotoxic activity against a wide variety of tumor cells in the absence of prior sensitization. Here we demonstrate that the integrin adhesion receptor LFA-1 (CD11a/CD18) regulates the cytotoxic activity of IL-12-activated NKT and NK cells against YAC-1 and EL-4 tumor cells. Differentiation in vivo and the expression of the cytolytic effector molecules perforin and Fas-L were comparable in both IL-12-activated NKT and NK cells from LFA-1-/ - and LFA-1+/+ mice. However, LFA-1-/-IL-12-activated NKT and NK cells showed impaired conjugate formation with target cells. These results provide the first genetic evidence for a role for an adhesion receptor in killing by IL-12-activated NK cells.     

Interleukin-15 enhances cytotoxicity, receptor expression, and expansion of neonatal natural killer cells in long-term culture

Newborn infants have a higher susceptibility to various pathogens due to developmental defects in their host defense system, including deficient natural killer (NK) cell function. In this study, the effects of interleukin-15 (IL-15) on neonatal NK cells was examined for up to 12 weeks in culture. The cytotoxicity of fresh neonatal mononuclear cells (MNC) as assayed by K562 cell killing is initially much less than that of adult MNC but increases more than eightfold after 2 weeks of culture with IL-15 to a level equivalent to that of adult cells. This high level of cytotoxicity was maintained for up to 12 weeks. In antibody-dependent cellular cytotoxicity (ADCC) assays using CEM cells coated with human immunodeficiency virus gp120 antigen, IL-15 greatly increased ADCC lysis by MNC from cord blood. IL-15 increased expression of the CD16+ CD56+ NK markers of cord MNC fivefold after 5 weeks of incubation. Cultures of neonatal MNC with IL-15 for up to 10 weeks resulted in a unique population of CD3- CD8+ CD56+ cells (more than 60%), which are not present in fresh cord MNC. These results show that IL-15 can stimulate neonatal NK cells and sustain their function for several weeks, which has implications for the clinical use of IL-15.     

Chemoattraction,adhesion and activation of natural killer cells are involved in the antitumor immune response induced by fractalkine/CX3CL1

Fractalkine (FK, also called neurotactin or CX3CL1) is a CX3C chemokine that can chemoattract T lymphocytes, monocytes, dendritic cells (DC) and natural killer (NK) cells. One of our previous studies demonstrated that FK in soluble form can chemoattract T cells and DC and membrane-bound FK can adhere T cells and DC. Vaccination with 3LL lung carcinoma cells gene-modified with FK (3LL-FK) induces potent antitumor CTL response. The aim of the present study is to investigate whether NK cells participate in FK-induced antitumor immunity. We found that NK activity was increased in mice inoculated with 3LL-FK and in vivo depletion of NK cells resulted in the decreased tumor growth inhibition of 3LL-FK, indicating that NK cells play an important role in the antitumor immunity induced by FK. Further studies showed 3LL-FK could chemoattract, adhere NK cells and attract more NK cells to infiltrate into tumor tissue. Incubation of NK cells with 3LL-FK could increase the cytotoxicity of NK cells against YAC-1 cells and even against NK-resistant parental 3LL cells. IL-12 production increased more significantly in the 3LL-FK tumor nodules. Taken together with CTL response induced by 3LL-FK, our data demonstrate that FK, expressed by gene-modified tumor cells, can induce potent antitumor effect through different mechanisms, one of which involves chemoattraction of NK cells into tumor sites and activation of NK cells.     

Natural antitumor defense system of the murine liver

Murine nonparenchymal liver cells from various genetic strains isolated by collagenase digestion and differential sedimentation contain both lymphocytes and macrophages. Nonparenchymal liver cells as well as spleen cells, mononuclear blood cells, and peritoneal exudate cells from C3HeB/FeJ mice were tested for natural cytotoxicity against YAC-1 (sensitive to NK cells) and P815 (resistant to NK cells) tumor cell lines. Resident peritoneal exudate cells exerted no cytotoxicity against either tumor cell, whereas spleen and mononuclear blood cells lysed only YAC-1. In contrast, nonparenchymal liver cells lysed both YAC-1(4 h) and P815 (18 h) tumor cells. Treatment of nonparenchymal liver cells with anti-asialo GM1 and complement abolished the antitumor activity against both tumor cell lines but not the phagocytic activity. Nonadherent nonparenchymal liver cells exerted greater cytotoxicity against YAC-1 tumor cells but little cytotoxicity against P815 tumor cells when compared with unfractionated cells. Adherent nonparenchymal liver cells (macrophages) from untreated mice exerted no antitumor activity against either tumor cell. In contrast, adherent nonparenchymal liver cells from Corynebacerium parvum treated mice were directly cytotoxic to P815 tumor cells. Spleen cells that are normally not cytotoxic to P815 tumor cells (18 h) became cytotoxic when mixed with adherent nonparenchymal liver cells from untreated mice. These results indicate that the tumoricidal effector cell in nonparenchymal liver cells from untreated mice appears to be the NK cell. Apparently, murine liver macrophages from untreated mice do not have tumoricidal activity per se but can "activate" NK cells to kill tumor cells normally resistant to NK cells.     

NKp46 defines a subset of bovine leukocytes with natural killer cell characteristics

Natural killer (NK) cells have not previously been precisely identified or characterized in cattle or any other ruminant species. We have generated a monoclonal antibody against bovine NKp46, which is expressed exclusively by NK cells in man. NKp46+ cells comprised 1-10% of blood mononuclear cells in cattle, and did not stain with antibodies against CD3, CD4, TCR1, B cell or granulocyte markers. The majority of the NKp46+ cells expressed CD2, and a variable fraction also expressed CD8. The tissue distribution of NKp46+ cells in cattle was compatible with the tissue distribution of NK cells in other species. Bovine NKp46+ cells had typical, large granular lymphocyte morphology, and proliferated vigorously in response to bovine IL-2 for a limited number of cell divisions. IL-2-activated NKp46+ cells killed the bovine kidney cell line MDBK. This cytotoxicity was inhibited by preincubation with antibody against NKp46. In a redirected lysis assay, IL-2-activated NKp46+ cells killed the FcgammaR+ target cell line P815 after preincubation with antibody against NKp46. Together, these data indicate that bovine NKp46 is anactivating receptor and demonstrate the existence of a subset of leukocytes in cattle that, in terms of surface markers, morphology and function, represent NK cells.     

Activation of human T cells with NK cell markers by staphylococcal enterotoxin A via IL-12 but not via IL-18

We have reported recently that mouse liver NK cells and NK1 x 1+ T cells were activated by bacterial superantigens via the IL-12 production from Kupffer cells. In the present study, we examined the effect of staphyloccoccal enterotoxin A (SEA) on human T cells with NK cell markers, CD56 or CD57 (NK-type T cells). After stimulating peripheral blood mononuclear cells (PBMC) with SEA, PBMC produced a large amount of IFN- and acquired a potent antitumour cytotoxicity. The in vitro depletion of either CD56+ TCR NK cells, CD56+ T cells or 57+ T cells from PBMC significantly inhibited the IFN- production from PBMC. When purified NK-type T cells, NK cells and regular T cells were cultured with monocytes and SEA they all produced IFN-, while the IFN- amounts produced by both NK-type T cells were greater than those produced by NK cells. NK cells as well as CD56+ T cells showed cytotoxicity against NK-sensitive K562 cells, whereas both NK-type T cells showed a more potent cytotoxicity against NK-resistant Raji cells than did NK cells. The IFN- production from each population as well as from whole PBMC was greatly inhibited by anti-IL-12 antibody but not by anti-IL-18 antibody. The antitumour cytotoxicity of whole PBMC was also significantly inhibited by anti-IL-12 antibody while the SEA-induced proliferation of PBMC was not affected by anti-IL-12 antibody. Furthermore, SEA-activated NK-type T cells as well as NK cells showed cytotoxicities against vascular endothelial cells. Our findings suggest that human NK-type T cells are thus involved in bacterial superantigen-induced immune response.     

Interleukin-15 Enhances Cytotoxicity, Receptor Expression, and Expansion of Neonatal Natural Killer Cells in Long-Term Culture

Newborn infants have a higher susceptibility to various pathogens due to developmental defects in their host defense system, including deficient natural killer (NK) cell function. In this study, the effects of interleukin-15 (IL-15) on neonatal NK cells was examined for up to 12 weeks in culture. The cytotoxicity of fresh neonatal mononuclear cells (MNC) as assayed by K562 cell killing is initially much less than that of adult MNC but increases more than eightfold after 2 weeks of culture with IL-15 to a level equivalent to that of adult cells. This high level of cytotoxicity was maintained for up to 12 weeks. In antibody-dependent cellular cytotoxicity (ADCC) assays using CEM cells coated with human immunodeficiency virus gp120 antigen, IL-15 greatly increased ADCC lysis by MNC from cord blood. IL-15 increased expression of the CD16⁺ CD56⁺ NK markers of cord MNC fivefold after 5 weeks of incubation. Cultures of neonatal MNC with IL-15 for up to 10 weeks resulted in a unique population of CD3⁻ CD8⁺ CD56⁺ cells (more than 60%), which are not present in fresh cord MNC. These results show that IL-15 can stimulate neonatal NK cells and sustain their function for several weeks, which has implications for the clinical use of IL-15.     

Natural killer cell mediated cytotoxicity of tumor cells initiated by neem leaf preparation is associated with CD40-CD40L-mediated endogenous production of interleukin-12

Neem leaf preparation (NLP) was found to activate natural killer (NK) cells (CD56(+)CD3(-)) to enhance their cytotoxic ability to tumor cells and stimulate the release of interleukin-12 (IL-12) from macrophages from healthy individuals and head-and-neck squamous cell carcinoma patients. NLP upregulated cytotoxic (CD16(+) and CD56(dim)) NK cells, and the cytotoxicity of NK-sensitive K562 cells by NLP-stimulated peripheral blood mononuclear cells decreased significantly after IL-12 neutralization. This NK-mediated cytotoxicity was manifest by upregulation of IL-12-dependent intracellular expression of the perforin-granzyme B system. Moreover, NK cytotoxic function was abolished after use of concanamycin A, a perforin inhibitor, but not by brefeldin A, a Fas inhibitor, confirming the participation of the perforin-granzyme B system. In addition NLP upregulated the expression of CD40 in CD14(+) monocytes and CD40L in CD56(+) lymphocytes. Neutralization of CD40 and CD40L in NLP-stimulated peripheral blood mononuclear cells culture resulted in significant downregulation of IL-12 release and cytotoxicity of NK cells, demonstrating the role of a CD40-CD40L interaction in the observed functions. Signals involved in the NLP-induced release of IL-12, and thereby induction of NK cell cytotoxicity, are mediated by activating p38MAPK pathway, but not through the ERK1/2 signaling pathway. Overall the results suggest that NLP effects NK cellular cytotoxicity by CD40-CD40L-mediated endogenous production of IL-12, which critically controls perforin-dependent tumor cell cytotoxicity.     

Changes of natural killer cell activity in different mouse lines by acute and asymptomatic flavivirus infections

Effect of certain flaviviruses on the activity of mouse natural killer (NK) cells was investigated using the classical mouse splenocyte system and YAC-1 cells for demonstration of NK cell cytotoxicity. Infection of mice with Langat and West Nile (WN) viruses was accompanied by temporary activation of NK cells. In mice infected with tick-borne encephalitis (TBE) virus the stimulation phase of NK cell cytotoxicity on days 2-4 post-infection (p.i.) was followed by suppression of their activity. As to the surface markers (sensitivity to antitheta and antiimmunoglobulin serum, respectively), the flavivirus-activated NK cells did not differ from the endogenous NK cells of intact mice. The stimulatory effect of flaviviruses on cytotoxicity of NK cells varied in different mouse lines. An increased NK cell activity at early stages of TBE virus infection was observed in mouse lines characterized by low (C57B1/6) and medium (BALB/c)--but not by high (CBA)-activity of their non-stimulated NK cells. Suppression of NK cell activity at later stages of TBE virus infection was not associated with virus multiplication in mouse splenocytes.     

Interleukin-12 induces cytotoxic NK1+ alpha beta T cells in the lungs of euthymic and athymic mice.

We recently reported that interleukin-12 (IL-12) stimulated hepatic NK1.1 Ag+ alpha beta T cells with intermediate T-cell receptor (TCR; NK1+ TCRint cells) and enhanced their NK1 expression (NK1high TCRint), and that these cells acquire strong major histocompatibility complex (MHC) unrestricted cytotoxicity in C57BL/6 mice, both +/+ and nu/nu. In the present study, we find that although murine lung normally has few NK1+ TCRint cells, NK1high TCRint cells are induced in+/+ and nu/nu mice after systemic administration of IL-12; these cells exhibit strong MHC unrestricted cytotoxicity against NK-sensitive and -resistant targets. A small number of NK1high TCRint cells was also found in peripheral blood after increased amounts of IL-12 were administered. Cytotoxicity tests in vitro revealed that the cytotoxic activity of the lung mononuclear cells (MNC) of C57BL/6 mice induced by IL-12 was abrogated by the depletion of either NK1+ or CD3+ cells, but not of CD8+ cells, as reportedly was the case of hepatic MNC, suggesting that NK1high TCRint cells are an antimetastatic population not only in the liver but also in the lung of mice. IL-12 injection into mice markedly elevates serum interferon-gamma (IFN-gamma) levels. However, although IL-12-induced cytotoxicity of NK1high TCRint cells was significantly reduced by anti-IFN-gamma antibody injection (which decreased serum IFN-gamma to an undetectable level), the appearance of NK1high TCRint cells in the lung and liver was not so affected. These results suggest that IFN-gamma is an important mediator of the cytotoxicity of NK1high TCRint cells but is not an essential factor for induction of these cells. We also added data showing that IL-12 has a broad antimetastatic effect against various liver and lung metastatic tumours intravenously injected into several strains of mice, including NK-deficient bg/bg mice. It can be considered that, in addition to NK cells, CD8+ cytotoxic T cells and gamma delta T cells, NK1+ TCRint cells can be categorized as one of the cytotoxic effector populations. These novel type cells distinct from regular T cells may play an important role in monitoring intra- and perivascular areas.     

The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1 Ag+ T cells in T helper 1 immune responses

Summary: The liver remains a hematopoietic organ after birth and can produce all leukocyte lineages from resident hematopoietic stem cells. Hepatocytes produce acute phase proteins and complement in bacterial infections. Liver Kupffer cells are activated by various bacterial stimuli, including bacterial lipopolysaccharide (LPS) and bacterial superantigens, and produce interleukin (IL)-12. IL-12 and other monokines (IL-18 etc.) produced by Kupffer cells activate liver natural killer (NK) cells and NK1.1 Ag+ T cells to produce interferon-g and thereby acquire cytotoxicity against tumors and microbe-infected cells. These liver leukocytes and the T helper 1 immune responses induced by them thus play a crucial role in the first line of defense against bacterial infections and hematogenous tumor metastases. However, if this defense system is inadequately activated, shock associated with multiple organ failure takes place. Activated liver NK1.1 Ag+ T cells and NK cells also cause hepatocyte injury. NK1.1 Ag+ T cells and another T‐cell subset with an intermediate T‐cell receptor, CD122+CD8+ T cells, can develop independently of thymic epithelial cells. Liver NK cells and NK1.1 Ag+ T cells physiologically develop in situ from their precursors, presumably due to bacterial antigens brought from the intestine via the portal vein. NK cells activated by bacterial superantigens or LPS are also probably involved in the vascular endothelial injury in Kawasaki disease.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.     

Interleukin-2 enhances the natural killer cell response to Herceptin-coated Her2/neu-positive breast cancer cells

The Her2/neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in 20% of breast adenocarcinomas and is recognized by a humanized anti-Her2/neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcgammaRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (IL-2) and become potent cytotoxic effectors following exposure to high doses of IL-2. We tested IL-2-activated NK cells against Her2/neu+ (MCF-7Her2/neu) and Her2/neu- (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2/neu and MDA-468 target cells by IL-2-activated NK cells was 35% and 3%, respectively (p < 0.05). Lysis was less than 5% when targets were treated with either the non-humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5-coated MCF-7Her2/neu cells was inhibited by 80 % when NK cells were pre-treated with an anti-Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF-7Her2/neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low-dose IL-2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5-coated tumor cells in the presence of IL-2 also produced large amounts of IFN-gamma with concomitant up-regulation of cell-surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL-2 therapy in patients with cancers that express the Her2/neu oncogene.     

Recognition and regulation of progenitor marrow elements by NK cells in the mouse.

The cytotoxicity of radiolabelled YAC-1 target cells by natural killer (NK) cells from the spleens of immunocompetent CBA mice is inhibited by unlabelled YAC-1 competitor cells, but not by resting bone marrow from syngeneic or allogeneic adult mice. Rapidly proliferating haemopoietic cells recovered from the spleens of lethally irradiated, bone marrow-reconstituted CBA mice, however, compete strongly in the NK assay. The competitive ability of early regenerating marrow correlates with the presence of an increased percentage of morphologically immature cells of mixed lineages. Competition declines in reconstituted spleens recovered more than 10 days after engraftment, as the proportion of immature elements falls towards that of resting marrow. Although the numbers of unlabelled YAC-1 cells required to produce equivalent competition of unstimulated and interferon-activated NK killing are similar, 10 times fewer regenerating marrow competitors compete cytotoxicity by unstimulated NK effectors to the same degree as interferon activated cells. The numbers of granulocyte-macrophage colonies formed in soft agar by regenerating marrow is also influenced by prior incubation of the marrow cells with NK effector populations. Spleen cells from homozygous athymic mice produce the same effect as cells from their heterozygous littermates. These data suggest that NK cells recognize and regulate the differentiation of progenitor elements within the marrow.     

The quantitative and functional changes of NK cells in mice infected with Angiostrongylus cantonensis

Angiostrongylus cantonensis is a neurotropic parasite which can cause injury to central nervous system and eosinophilic meningitis to human. Natural killer (NK) cells are specialized innate lymphocytes important in early defense against pathogens as in a variety of intracellular bacterial, viral, and protozoan infections. However, the number and function of NK cells in extracellular parasitic infection of A. cantonensis are unclear. In this study, on A. cantonensis infected mice which may mimic the human’s infection, we found that the percentage of splenic NK cells and the absolute number of peripheral blood NK cells were decreased at 21-day post infection compared with that of controls. When administrating with albendazole treatment at early stage of the infection, the changes of NK cells could be avoided. Further analysis confirmed that the reduction of NK cells was due to their apoptosis manifested as increased expressions of annexin V and activated caspase-3 after 16-day post infection. Moreover, both activated and inhibitory receptors such as CD16, CD69, NKG2D, and Ly49a on NK cells were down-regulated after 16-day post infection. Interestingly, NK cells isolated from mice of 21-day post infection showed enhanced IFN-γ production when stimulated with IL-12 for 24 h and cytotoxicity to YAC-1 cells, as well as elevated CD107a expression. It is evident that NK cell population and its function were changed in A. cantonensis infected mice, suggesting their involvement in pathogenesis of the infection.