Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.     

Murine natural killer cell differentiation: past, present, and future

Natural killer cells are bone marrow-derived lymphocytes capable of lysing a variety of target cells without prior exposure. While the biological activities and function of mature NK cells have been extensively investigated, the differentiation of NK cells from primitive hematopoietic stem cells is poorly understood. Recently, we have reported on the identification of a highly enriched bone marrow population capable of repopulating recipient mice with mature NK cells. In this review, we will summarize our findings and those of others in an attempt to clarify the current status of murine natural killer cell differentiation.     

Expression of killer cell inhibitory receptors on human uterine natural killer cells

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer (NK) cells within the maternal decidua in close proximity to the fetally-derived invading extravillous trophoblast which expresses at least two HLA class I molecules, HLA-G and HLA-C. These NK cells have an unusual phenotype, CD56(bright) CD16, distinguishing them from adult peripheral blood NK cells. They may control key events in trophoblast migration and therefore placentation. Human NK cells in peripheral blood express receptors for polymorphic HLA class I molecules. This family of receptors, known as killer cell inhibitory receptors (KIR), are expressed on overlapping subsets of NK cells to give an NK cell repertoire which differs between individuals. Using a panel of monoclonal antibodies to several members of the KIR family and analysis by flow cytometry, we have found that KIR are expressed by decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. Comparison of NK cells from decidua and peripheral blood of the same individual showed that NK cells from these two different locations express different repertoires of KIR. Receptors are present in individuals who do not possess the relevant class I ligand, raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site.     

Human natural killer cells

Human natural killer (NK) cells comprise 10 to 15% of peripheral blood lymphocytes, characterized by their morphologic appearance of large granular lymphocytes (LGLs) and phenotype CD3- CD56+ CD16+ or CD16-. Functionally, these cells are defined by their ability to lyse target cells without prior sensitization and without restriction by major histocompatibility (MHC) antigens. These cells play an important role in immune defenses, especially after hematopoietic transplantation. They contribute to the defenses against virus-infected cells, graft rejection, and neoplasias; they also participate in the regulation of hematopoiesis through cytokine production and cell to cell interaction. In this mini-review we attempt to summarize the most relevant findings about these cells in terms of their origin and differentiation, their cell surface characteristics including activation and their cytolytic pathways. We have also provided a brief approach of their potential clinical use. Increased knowledge of NK cell differentiation, ontogeny and regulatory mechanisms may be of use for the planning of immunotherapeutic strategies.     

Natural killer activating receptors trigger interferon gamma secretion from T cells and natural killer cells

Proliferation of human CD4+ alphabeta T cells expressing a natural killer cell activating receptor (NKAR) has been shown to be enhanced, particularly in response to low doses of antigen, if the target cells present appropriate human class I major histocompatibility complex (MHC) molecules. Here, we show that NKAR also enhance proliferation and killing of target cells by subsets of CD8+ alphabeta and CD8+ gammadelta T cells, as well as by NK cells. Strikingly, interferon gamma secretion from all of these types of lymphocytes was markedly increased by interaction of the NKAR with their MHC class I ligands, independently of enhancement of proliferation. Thus, the recognition of class I MHC molecules by NKAR on both T cells and NK cells may provide a regulatory mechanism that affects immune responses through the secretion of interferon gamma and possibly other cytokines. It represents a signal for cytokine secretion alternative and/or augmentative to that through the T cell receptor.     

Function and specificity of human natural killer cell receptors

In humans, natural killer lymphocytes express HLA class I-specific inhibitory receptors belonging to at least two different molecular families. The first is represented by members of the Ig superfamily that are involved in the recognition of different groups of HLA class I alleles, and the second is represented by a molecular complex formed by CD94 and NKG2A that displays a broad specificity for various class I molecules including the 'non-classical' HLA-G molecules. In addition to the inhibitory receptors, a series of activating receptors has been identified. Some display the same specificities as the corresponding inhibiting receptors and can be viewed as HLA class I-specific activating receptors. Another group of activating receptors appear to be involved in the cytolytic activity against HLA-'negative' target cells. These receptors are clearly non-MHC specific and, under physiological conditions, their function is suppressed by the HLA class I-specific inhibitory receptors.     

Inhibition of natural killer cell cytotoxicity by cell growth-related molecules

Certain MHC class I molecules on target cells are known to inhibit the cytotoxic action of NK cells. By using monoclonal antibody (mAb) Cho-1, we have found inhibitory non-MHC class I cell surface molecules that are noncovalently-associated with 200 kDa and 40 kDa antigens. Poly I-C-induced rat NK cells were not cytotoxic to rat fetus-derived fibroblast WFB cell line. In contrast, NK cells were cytotoxic to H-ras oncogene-induced transformants of WFB, W14 and W31. FACS analysis indicated that mAb Cho-1 reacts with WFB, but not with W14 and W31 cells. Thus, this antigen may disappear concomitantly with cell growth and transformation. Cho-1 antigens were also expressed on other NK-resistant lines, such as mouse BALB3T3 fibroblast, EL-4 lymphoma and human fibroblast HEPM. However, they were not expressed on NK-sensitive mouse YAC-1 and H-ras transformant (Brash) of BALB3T3 cells. Furthermore, treatment of target cells with IFN-gamma clearly induced the cell surface expression of Cho-1 antigens, and conferred a resistance to NK cytolysis on target cells. These data strongly suggest that Cho-1 antigen expression may correlate with target cell susceptibility to NK cells. Indeed, treatment of NK-resistant WFB as well as HEPM cells with F(ab')2 fragments of mAb Cho-1 resulted in the acquisition of susceptibility to NK cytolysis. Cho-1 antigens may be novel molecules that regulate the NK resistance of cells.     

Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity

Natural killer (NK) cells take up chylomicrons (CM), very low density (VLDL), low density (LDL), high density (HDL) and acetyl-modified low density (AcLDL) lipoproteins through different receptors, VLDL being the lipoprotein with the highest uptake and HDL the lowest. The uptake of LDL can be selectively blocked by the anti-LDL receptor, which does not affect the uptake of CM, VLDL, HDL and AcLDL. Although the uptake of lipoproteins assessed by flow cytometry using DiI is not very high, the lipoproteins are able to induce an increase in proliferative responses, VLDL, AcLDL and HDL being the most important ones with 12- and 17-fold increments, respectively. CM, VLDL and LDL at low concentrations increase NK cytotoxic activity, while HDL and AcLDL inhibit, in a dose-dependent fashion, the killing of NK cells against K562. These results suggest the presence of four different receptors that are responsible for the cytotoxic and proliferative responses observed.     

Receptors that turn on and turn off natural killer cells

The receptors on natural killer cells for polymorphic major histocompatibility complex or human leukocyte antigen class I molecules have recently been cloned. Two structurally distinct receptor families have been identified from murine and human natural killer cells. These receptors are classified as members of the C-type lectin and immunoglobulin families. Initially, it appeared that murine natural killer cells express the C-type lectin receptors whereas human natural killer cells express immunoglobulin-like receptors. Recent data suggest that human natural killer cells can express both C-type lectin-like receptors and the immunoglobulin-like receptors. The interaction of natural killer-cell receptors with class I molecules was characterized to inhibit natural killer-cell activation; however, allelic forms of these natural killer cell receptors have recently been identified to also have activating properties. These discoveries show the presence of a diversity of receptors for major histocompatibility complex class I molecules on T and natural killer cells. In addition they illustrate the use of two unique strategies by the immune system for eliminating altered- or non-self cells: through the detection of foreign antigens and through the detection of absence of self antigens.     

Increased sensitivity of gastric cancer cells to natural killer and lymphokine-activated killer cells by antisense suppression of N-acetylgalactosaminyltransferase

UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) catalyze the initial reaction in O-linked (mucin type) oligosaccharide biosynthesis. To attempt to inhibit the synthesis of O-glycan, we transfected antisense cDNA of GalNAc transferase type 1 (GalNAc-T1) into a human gastric cancer cell line, JRST. A decreased expression of GalNAc-T1 at the level of both mRNA and protein was observed in the resultant transfectants. They demonstrated a significantly increased sensitivity to NK and lymphokine-activated killer cells in vitro compared with parental cells and mock transfectants. Although there was no significant difference in in vitro cell proliferation among parental cells, mock transfectants, or antisense transfectants, the in vivo growth rate of antisense transfectants using SCID mice was clearly lower than that of parental cells and mock transfectants. Furthermore, the treatment of mice with anti-asialo-G(M1) Ab abolished this growth-inhibitory effect of antisense transfection. From these results, we conclude that antisense suppression of GalNAc-T1 could increase the sensitivity of tumor cells to NK and lymphokine-activated killer cells, suggesting that this strategy may be of use as a new immunogene therapy.     

Anesthesia inhibits interferon-induced natural killer cell cytotoxicity via induction of CD8+ suppressor cells

Anesthesia (Avertin, halothane, isoflurane, ether, or ketamine/xylazan) of mice inhibits subsequent stimulation of splenic natural killer cell (NK) cytotoxicity by interferon (IFN) treatment either in vitro and in vivo. The current data demonstrate (a) in vitro depletion of CD8+ cells from mononuclear splenocytes of anesthetized mice restored the ability of NK cells to respond in vitro to IFN stimulation and (b) coincubation of CD8+ splenocytes from anesthetized mice with CD8- splenocytes of naive mice resulted in a significant reduction of the IFN-induced stimulation of NK activity in the coculture. These results suggest that anesthesia induces CD8+ cells that suppress stimulation of NK cytotoxicity by IFN.     

Effect of eccentric exercise on natural killer cell activity

The effect of eccentric one-legged exercise on natural killer (NK) cell activity was studied in eight healthy males. To distinguish between local and systemic effects, blood samples were collected from veins in the exercising leg and resting arm. However, the results did not significantly differ between the leg and arm. To eliminate diurnal variations, the results were compared with a control group that did not exercise but had blood samples collected at the same time points. In the exercising group, plasma creatine kinase increased progressively during and up to 4 days after exercise. The percentage of CD16+ NK cells increased during exercise, which was paralleled by an increase in the NK cell activity per fixed number of blood mononuclear cells. The NK cell activity on a per NK cell basis did not change. The percentage of CD3+, CD4+, CD8+, CD19+, and CD14+ cells did not change significantly during exercise. The present study thus showed that eccentric exercise with a relatively small muscle mass (1 quadriceps femoris muscle) causes systemic effects on NK cells. It is suggested that the increase in plasma epinephrine during eccentric exercise is responsible for the observed increase in the percentage of CD16+ cells.     

Migratory response of human natural killer cells to lymphotactin

Lymphotactin (Lptn) is a new protein belonging to the C or gamma subfamily of chemokines with only two of the four cysteine residues. Lptn was reported to act specifically on T lymphocytes and not on monocytes and neutrophils. To understand better the spectrum of action of Lptn we have examined its ability to induce natural killer (NK) cell migration. Freshly isolated human NK cells as well as long-term cultured NK cells propagated in interleukin-2 (IL-2)-containing medium migrated in response to Lptn. Optimal activity was observed at concentrations ranging from 50 to 200 ng/ml, and the efficacy was comparable to that of MCP-1, the prototype of C-C chemokines. Migration in response to Lptn was chemotaxis rather than chemokinesis as determined in a checkerboard analysis. Migration of NK cells was comparable to that observed with T lymphocytes from the same donor, under the same experimental conditions. Finally, in contrast to other cytokines (IL-2 and IL-12) which in addition to chemotaxis augment NK cell adhesion to endothelial cells in vitro, Lptn did not affect the adhesiveness of NK cells to vascular endothelium.     

Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells

Fas ligand (FasL) triggers apoptosis during cytotoxicity mediated by cytotoxic T lymphocytes and during immune downregulation. The ability of T cells and natural killer cells to trigger apoptosis through this mechanism is controlled by the cell surface expression of FasL (ref. 2). Because FasL expression is up-regulated on activation, FasL was thought to be delivered directly to the cell surface. Here we show that newly synthesized FasL is stored in specialized secretory lysosomes in both CD4+ and CD8+ T cells and natural killer cells, and that polarized degranulation controls the delivery of FasL to the cell surface. In this way, FasL-mediated apoptosis is finely controlled by receptor-mediated target-cell recognition. The cytoplasmic tail of FasL contains signals that sort FasL to secretory lysosomes in hemopoietic cells. This pathway may provide a general mechanism for controlling the cell surface appearance of proteins involved in immune regulation.     

A boy with X-linked hyper-IgM syndrome and natural killer cell deficiency

OBJECTIVE: To compare the efficacy of nifedipine with ritodrine in the management of preterm labor. METHODS: One hundred eighty-five singleton pregnancies with preterm labor were assigned randomly to either ritodrine intravenously (n = 90) or nifedipine orally (n = 95). The principal outcome assessed was delay of delivery. RESULTS: Ritodrine was discontinued in 12 patients because of severe maternal side effects, and their results were excluded from further analysis. More women in the ritodrine group delivered within 24 hours (22 versus 11, P = .006), within 48 hours (29 versus 21, P = .03), within 1 week (45 versus 36, P = .009), and within 2 weeks (52 versus 43, P = .005) compared with those receiving nifedipine. There were significantly fewer maternal side effects in the nifedipine group. Apgar scores and umbilical artery and vein pHs were similar in both groups. The number of admissions to the neonatal intensive care unit (NICU) in the nifedipine group was significantly lower than in the ritodrine group (68.4 versus 82.1%, P = .04). CONCLUSION: Nifedipine in comparison with ritodrine in the management of preterm labor is significantly associated with a longer postponement of deliver, fewer maternal side effects, and fewer admissions to the NICU.     

Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium

Hematopoietic stem cells are capable of extensive self-renewal and expansion, particularly during embryonic growth. Although the molecular mechanisms involved with stem cell maintenance remain mysterious, it is now clear that an intraembryonic location, the aorta-gonad-mesonephros (AGM) region, is a site of residence and, potentially, amplification of the definitive hematopoietic stem cells that eventually seed the fetal liver and adult bone marrow. Because several studies suggested that morphologically defined hematopoietic stem/progenitor cells in the AGM region appeared to be attached in clusters to the ventrally located endothelium of the dorsal aorta, we derived cell lines from this intraembryonic site using an anti-CD34 antibody to select endothelial cells. Analysis of two different AGM-derived CD34(+) cell lines revealed that one, DAS 104-8, efficiently induced fetal-liver hematopoietic stem cells to differentiate down erythroid, myeloid, and B-lymphoid pathways, but it did not mediate self-renewal of these pluripotent cells. In contrast, a second cell line, DAS 104-4, was relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early hematopoietic progenitor cells of the lin-CD34(+)Sca-1(+)c-Kit+ phenotype and was proficient at maintaining fetal liver-derived hematopoietic stem cells able to competitively repopulate the bone marrow of lethally irradiated mice. These data bolster the hypothesis that the endothelium of the AGM region acts to mediate the support and differentiation of hematopoietic stem cells in vivo.     

Human natural killer cell committed thymocytes and their relation to the T cell lineage

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway.     

Enhancement of megakaryocytopoiesis by Campath-1G-treated natural killer cells

OBJECTIVE: To assess the effect of vertical banded gastroplasty and gastric banding on the development of gastro-oesophageal reflux using both subjective and objective methods. DESIGN: Prospective, randomised study. SETTING: Teaching hospital, Sweden. SUBJECTS: 50 consecutive, morbidly obese patients (mean (SD) body mass index (BMI) 42.5 (5), 27 women and 23 men; mean age 48 years, range 38-57 years). INTERVENTIONS: Vertical banded gastroplasty (n = 24) or gastric banding (n = 26). MAIN OUTCOME MEASURES: Results of evaluation by standardised questionnaire, 24-hour ambulatory pH-metry, and endoscopy. RESULTS: After six months the mean (SD) BMI had decreased to 34.4 (5.7), with no differences between the groups. Mild dysphagia was somewhat more common (13 compared with 1) but the incidence of heartburn (8 compared with 17), and acid regurgitation (12 compared with 14) were less after the operation; 3 patients developed erosive oesophagitis, two in the vertical banded group and one in the banding group. Ambulatory pH-metry readings did not change significantly from preoperatively and there were no differences between the two operations. One patient developed pathological reflux, and in three the values returned to the normal range. CONCLUSION: Gastric restriction operations including those with a narrow stoma that causes outflow obstruction do not seem to increase the incidence of reflux in patients with functioning antireflux barriers.