In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

The effect of radiation therapy combined with natural killer cells against spontaneous murine fibrosarcoma

The effect of radiation therapy combined with lymphoid cells against spontaneous murine fibrosarcoma (FSa-II) was investigated bothin vivo andin vitro. In thein vivo experiment, syngeneic C3H mice were divided into 3 groups. Animals in the first group were injected with 1 x 10⁵ tumor cells into the right hind leg. Animals in the second and third groups were injected with 1 x 10⁵ tumor cells mixed with 1 x 10⁷ normal lymphoid cells (NLC) or effector lymphoid cells (ELC), respectively. ELC were obtained from spleen and lymph nodes of FSa-II-bearing mice and incubatedin vitro for 40 hr to eliminate suppressor T cell function. NLC were obtained from normal mice and incubated in the same way. Irradiation was given using¹³⁷Cs unit 3 days after cell inoculation. 12 out of 14 mice (85.7%) inoculated with tumor cells mixed with NLC did not show any tumor growth at 60 Gy local irradiation. 12 out of 21 mice (57.1 %) inoculated with tumor cells alone and 6 out of 10 (60%) with tumor cells mixed with ELC rejected tumors at the same radiation dose. This synergistic effect with NLC was not observed when NLC was inoculated after irradiation, indicating that lymphoid cells should be in contact with tumor cells before irradiation. In the⁵¹Cr release assay, lymphoid cells obtained from whole body irradiated (WBI) mice showed 17.8% lysis without irradiation and 28.8% lysis at 5 Gy irradiation. Untreated NLC showed almost no cytotoxic effect at the same radiation dose. This synergistic effect disappeared when WBI lymphoid cells were treated with anti asialo GM1 and complement. These results suggested that NK cells might be important in this synergistic effect with irradiation. To obtain a sufficient level of synergistic effect by in vitro combined treatment of mixed tumor cell - NLC culture and irradiation - incubation for more than 12 hrs and 8 hrs appeared to be necessary before and after irradiation, respectively.     

Trafficking of natural killer cells

Natural killer (NK) cells comprise a set of lymphocytes that is capable of mediating innate immune responses to viral infections, malignancies, and allogeneic bone marrow grafts. This review summarizes what is known about the mechanisms NK cells use to arrive at their sites of action. NK cells express a wide array of adhesion molecules including alphaLbeta2, alphaMbeta2, alphaXbeta2, and alpha4beta1 integrins, ICAM-1, PSGL-1, and L-selectin. Like other immune and inflammatory cells, NK cells use the blood circulation to enter tissues and organs, which requires that they interact with the vessel wall under flow conditions, arrest, and transmigrate. NK cells are able to chemotax to a variety of cytokines and chemokines, including IL-12, IFN-(alpha/beta, CCL2, 3, 4, 5, 7, 8, CXCL8, and CX3CL1. In many cases, NK cells appear to migrate towards these soluble factors without any kind of priming. These cells also appear to distribute in secondary and tertiary lymphoid sites (i.e., spleen, bone marrow, liver, lung, and lymph nodes) both with and without stimulation. In addition to their ability to move throughout the body in an unprimed state, activated NK cells may have increased specificity in homing to sites of inflammation. NK cells not only react to, but also produce IFN-gamma, TNF-alpha, GM-CSF, CCL3, CCL4, and CCL5, enabling them to recruit various immune cells to sites of immune response.     

Immunoregulation by natural killer cells

Polyinosinic-polycytidilic acid (poly (I:C], a synthetic analog of viral double-stranded RNA (dsRNA), activates natural killer (NK) cells and inhibits induction or promotes termination of the primary IgM response in vivo. Suppression of responses was reproduced in vivo by interferons (IFN) which activate NK cells and in vitro by cells enriched for NK cells. The likelihood that NK cells may be involved in the normal regulation of IgM responses is supported by the following observations: immunization itself induces NK activity at times appropriate to account for termination, NK cells activated by immunization suppress in vitro, mice with high NK activity induced by immunization with one antigen have reduced responses to immunization with a second antigen, and mice with induced loss of NK activity fail to down-regulate IgM antibody responses normally.     

Self-tolerance of natural killer cells

Natural killer (NK) cells, similar to other lymphocytes, acquire tolerance to self. This means that NK cells have the potential to attack normal self cells but that there are mechanisms to ensure that this does not usually occur. Self-tolerance is acquired by NK cells during their development, but the underlying molecular and cellular mechanisms remain poorly understood. Recent studies have produced important new information about NK-cell self-tolerance. Here, we review the evidence for and against possible mechanisms of NK-cell self-tolerance, with an emphasis on the role of MHC-specific receptors.     

Homeostasis of natural killer cells

Natural killer (NK) cells are important players of innate immunity, dedicated to the host defense against viruses and also involved in the immune surveillance of tumors. NK cells are widely distributed in the body and their number may increase locally during infection. They develop mainly in the bone marrow and perhaps in other lymphoid organs. They are constantly renewed, with a half-life of about 17 days at the periphery. In this article, we review the factors that regulate the homeostasis of NK cells including their development, differentiation, export to the periphery, their turnover, their homeostatic or antigen-induced proliferation and their survival before or after activation. In addition, we discuss the homeostasis of recently described so-called "memory" NK cells.     

Immunotherapy with ligands of natural killer T cells

Natural killer T (NKT) cells are innate lymphocytes that share receptor structures and functions with conventional T cells and natural killer cells. NKT cells are specific for glycolipid antigens bound by the major histocompatibility complex class I-like protein CD1d. One striking property of NKT cells is their capacity to rapidly produce large amounts of cytokines in response to T-cell receptor engagement, suggesting that activated NKT cells can modulate adaptive immune responses. Recent pre-clinical studies have revealed significant efficacy of NKT-cell ligands such as the glycolipid alpha-galactosylceramide for treatment of metastatic cancers and infections, and for prevention of autoimmune diseases. These findings suggest that appropriate stimulation of NKT cells could be exploited for prevention or treatment of human diseases.     

Influence of cytokines on HIV-specific antibody-dependent cellular cytotoxicity activation profile of natural killer cells

There is growing interest in HIV-specific antibody-dependent cellular cytotoxicity (ADCC) as an effective immune response to prevent or control HIV infection. ADCC relies on innate immune effector cells, particularly NK cells, to mediate control of virus-infected cells. The activation of NK cells (i.e., expression of cytokines and/or degranulation) by ADCC antibodies in serum is likely subject to the influence of other factors that are also present. We observed that the HIV-specific ADCC antibodies, within serum samples from a panel of HIV-infected individuals induced divergent activation profiles of NK cells from the same donor. Some serum samples primarily induced NK cell cytokine expression (i.e., IFNγ), some primarily initiated NK cell expression of a degranulation marker (CD107a) and others initiated a similar magnitude of responses across both effector functions. We therefore evaluated a number of HIV-relevant soluble factors for their influence on the activation of NK cells by HIV-specific ADCC antibodies. Key findings were that the cytokines IL-15 and IL-10 consistently enhanced the ability of NK cells to respond to HIV-specific ADCC antibodies. Furthermore, IL-15 was demonstrated to potently activate "educated" KIR3DL1(+) NK cells from individuals carrying its HLA-Bw4 ligand. The cytokine was also demonstrated to activate "uneducated" KIR3DL1(+) NK cells from HLA-Bw6 homozygotes, but to a lesser extent. Our results show that cytokines influence the ability of NK cells to respond to ADCC antibodies in vitro. Manipulating the immunological environment to enhance the potency of NK cell-mediated HIV-specific ADCC effector functions could be a promising immunotherapy or vaccine strategy.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

Carbohydrates in the functions of natural killer cells.

There is little doubt that carbohydrates are crucial to the functions of NK cells. Exogenous carbohydrates alter the cytolytic capabilities of these cells, treatment of NK cells with glycosidases or inhibitors of glycosylation affect function, NK cells can bind selective sugars, and NK cells can be identified and subdivided into functionally distinct subsets on the basis of cell surface carbohydrates. Yet, despite the large number of observations which have been made concerning carbohydrates and NK cells, there is little consensus regarding these studies and few investigations which satisfactorily demonstrate specific mechanisms for the observed effect of the carbohydrates. Almost certainly, the number of diverse observations implies roles for carbohydrates in multiple areas of NK function, probably including target recognition, tissue distribution and post-binding events in the lytic cascade. Hopefully, these observations made to date will be viewed as exciting preliminary studies which will entice more sophisticated investigations designed to elucidate the precise roles of carbohydrates in the functions of NK cells.     

Delayed hypersensitivity and nonspecific cellular immunity. The cytotoxic activity of macrophages, natural and antibody-dependent killer cells

The experiment on (BALB/cXC57BL)F1 mice, showing a high level of delayed hypersensitivity (DH) when sensitized with BCG vaccine and Staphylococcus aureus strain B-243, has demonstrated the influence of such sensitization and DH reaction induced by the injection of a specific antigen (old tuberculin or staphylococcal phagolysate) into the sensitized animals on the cytotoxicity of macrophages, natural killers (NK) and antibody-dependent killers (ADK). Sensitization with BCG vaccine alone results in an insignificant rise in the activity of these effector cells, and sensitization with S. aureus produces no changes at all. The pronounced activation of the cytotoxicity of macrophages, NK and, to a lesser extent, ADK has been observed in DH reaction induced by the injection of a specific antigen into the sensitized mice. In the course of DH reaction a rise in the activity of NK and ADK not only against tumor target cells, but also against microbial ones (Candida albicans and S. aureus) has been found to occur.     

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1×10⁻⁷ M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1×10⁻⁷ M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear³H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of Il-2 through its ability to enhance the expression of intermediate affinity Il-2R on these cells.     

Cancer immunotherapy with interleukin-2-activated natural killer cells

Natural killer (NK) cells are a subset of lymphocytes with a distinct morphologic appearance (large granular lymphocytes [LGL]) and the ability to kill virally infected and tumor targets but to spare most normal cells. NK cells respond to a variety of biologic agents, such as interleukin-2 (IL-2), or interferons, by upregulation of cytolytic, secretory, and proliferative functions. In cancer-bearing hosts, NK cells have been considered to be the major component of antitumor immunity responsible for rapid elimination of malignant cells from the blood. More recently, however, studies have demonstrated the ability of adoptively transferred, IL-2-activated NK cells to selectively localize into solid tumors tissue and to eliminate established tumors. While these findings indicate a role for NK cells in cancer immunotherapy, additional studies are needed in both animal models and in humans to optimize clinical protocols of cancer therapy based on these cells.     

Organ-specific features of natural killer cells

Natural killer (NK) cells can be swiftly mobilized by danger signals and are among the earliest arrivals at target organs of disease. However, the role of NK cells in mounting inflammatory responses is often complex and sometimes paradoxical. Here, we examine the divergent phenotypic and functional features of NK cells, as deduced largely from experimental mouse models of pathophysiological responses in the liver, mucosal tissues, uterus, pancreas, joints and brain. Moreover, we discuss how organ-specific factors, the local microenvironment and unique cellular interactions may influence the organ-specific properties of NK cells.     

Suicide behavior of target cells after binding with natural killer cells

Human natural killer (NK) cell activity seems to be related to the integrity and function of the cytoskeletal apparatus. It has been hypothesized that microfilaments and microtubules play a pivotal role. In particular, the binding of the NK cell to the target cell requires microfilament integrity, and the lysis of bound targets seems to depend on microtubule assembly. We focused on the changes occurring in cytoskeletal elements and surface structures of NK cells and of target cells highly sensitive to NK activity (K562). Our observations, performed by fluorescence and scanning electron microscopy, besides confirming a rearrangement of the cytoskeletal apparatus in the effector cell, provide evidence that target cell cytoskeletal elements are involved in NK cell function. In K562 cells, after binding with NK cells, there is marginal rearrangement of actin and polarization of tubulin and vimentin in the contact regions, accompanied by modification of surface structures. These findings suggest that the target cell plays an active role in its own death by participating in the formation of an extended area of intimate contact with the killer cell. In addition, they lend credence to the surprising proposal that NK cells may induce a suicide mechanism in target cells.     

Renewal of natural killer cells in mice having elevated natural killer cell activity

The present study was designed to examined the dynamics of splenic natural killer (NK) cells under two conditions of enhanced NK cell activity: (1) CBA/J mice given polyinosinic-polycytidylic acid (poly-I:C), an NK-cell-enhancing agent, and 62) untreated athymic nude (nu/nu) mice. The 'total NK cell activity' of the spleen (percentage specific lysis corrected for changes in organ cellularity) increased 5-fold and 2.7-fold after poly-I:C treatment for 1 day and 4 days, respectively. An injection of hydroxyurea (HU), a cell-cycle-toxic drug, given together with either poly-I:C or saline to CBA/J mice resulted in both cases in a 25% reduction in total NK cell activity 1 day later. This suggests that the renewal rate of nondividing NK cells is similar in poly-I:C-treated and saline-injected mice, and that the NK-enhancing effect of poly-I:C is not due to a stimulation of proliferation among NK cell precursors. HU administered simultaneously with poly-I:C or saline for 4 days eliminated NK cell activity in both cases, indicating that spleen NK cell activity is mediated almost entirely by newly formed (less than or equal to 4 days) cells. In nude mice, NK cell activity was assayed at various intervals after an HU depletion period of 2 days. NK depletion was initially more rapid in nu/nu mice than in control (nu/+) mice, although equally profound, and the subsequent recovery of NK cell activity after cessation of HU was also more rapid than in control (nu/+) mice.(ABSTRACT TRUNCATED AT 250 WORDS)