Recognition of virus infected cells by NK cells

NK cells show cytotoxicity against virus-infected cells and tumor cells and play an important role in host defense. Although mecheanism of target cell recognition by NK cells have been unclear for a long time, it has recently been elucidated that certain NK cell receptors specifically recognize virus products. Furthermore, expression pattern of NK cell receptors, which consist of activating and inhibitory receptors, determines susceptibility to virus-infection. Here, we review recent progress of mechanism of recognition of virus-infected by NK cells.     

Recognition of vaccinia virus-infected cells by human natural killer cells depends on natural cytotoxicity receptors

Natural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.     

Inability of interferon to protect virus-infected cells against lysis by natural killer (NK) cells correlates with NK cell-mediated antiviral effects in vivo

Murine cytomegalovirus (MCMV) is a natural killer (NK) cell-sensitive virus, whereas lymphocytic choriomeningitis virus (LCMV) is an NK cell-resistant virus. Selective depletion of NK cell activity by injection of mice with anti-asialo GM1 antibody enhanced synthesis of MCMV but not that of LCMV when mice were simultaneously infected with the two viruses. This suggests that the NK cell-mediated antiviral effects may depend on target cell susceptibility to NK cell-mediated lysis rather than the ability of a virus to induce a specialized antiviral NK cell. In support of this concept, activated NK cells isolated from either MCMV- or LCMV-infected mice had similar patterns of killing against all targets tested. Mouse embryonic fibroblasts (MEF) infected with MCMV were less sensitive to lysis by activated NK cells than either uninfected or LCMV-infected MEF. However, when MEF were pretreated with IFN, activated NK cell-mediated lysis against MCMV-infected MEF was undiminished and was much higher (up to fourfold) than that against uninfected MEF, whose sensitivity to lysis was almost totally abolished by IFN pretreatment. LCMV-infected MEF were also protected by IFN against activated NK cell-mediated lysis. During infection, the virus-induced IFN may protect uninfected and LCMV-infected cells from IFN-activated, NK cell-mediated lysis, but MCMV-infected cells may remain sensitive to lysis. This could explain how NK cells play a role in resistance to MCMV but not LCMV.     

The inhibitory receptor NKG2A determines lysis of vaccinia virus-infected autologous targets by NK cells

Signals transduced by inhibitory receptors that recognize self-MHC class I molecules prevent NK cells from being activated by autologous healthy target cells. In order for NK cells to be activated upon contact with an infected cell, the balance between the activating and inhibitory signals that regulate NK cell function must be altered in favor of activation. By studying liver-derived NK cells, we show that only a subpopulation of NK cells expressing high levels of the inhibitory receptor NKG2A are able to lyse autologous vaccinia-infected targets, and that this is due to selective down-regulation of HLA-E. These data demonstrate that release from an inhibitory receptor:ligand interaction is one mechanism that permits NK cell recognition of a virally infected target, and that the variegated expression of inhibitory receptors in humans generates a repertoire of NK cells with different antiviral potentials.     

Analysis of virus-infected cells by flow cytometry

Flow cytometry has been used to study virus-cell interactions for many years. This article critically reviews a number of reports on the use of flow cytometry for the detection of virus-infected cells directly in clinical samples and in virus-infected cultured cells. Examples are presented of the use of flow cytometry to screen antiviral drugs against human immunodeficiency virus (HIV), human cytomegalovirus, and herpes simplex viruses (HSV) and to perform drug susceptibility testing for these viruses. The use of reporter genes such as green fluorescent protein incorporated into HIV or HSV or into cells for the detection of the presence of virus, for drug susceptibility assay, and for viral pathogenesis is also covered. Finally, studies on the use of flow cytometry for studying the effect of virus infection on apoptosis and the cell cycle are summarized. It is hoped that this article will give the reader some understanding of the great potential of this technology for studying virus cell interactions.     

Turnover rates of B cells,T cells,and NK cells in simian immunodeficiency virus-infected and uninfected rhesus macaques

We determined average cellular turnover rates by fitting mathematical models to 5-bromo-2'-deoxyuridine measurements in SIV-infected and uninfected rhesus macaques. The daily turnover rates of CD4(+) T cells, CD4(-) T cells, CD20(+) B cells, and CD16(+) NK cells in normal uninfected rhesus macaques were 1, 1, 2, and 2%, respectively. Daily turnover rates of CD45RA(-) memory T cells were 1%, and those of CD45RA(+) naive T cells were 0.5% for CD4(+) T cells and approximately 1% for CD4(-)CD45RA(+) T cells. In SIV-infected monkeys with high viral loads, the turnover rates of T cells were increased approximately 2-fold, and that of memory T cells approximately 3-fold. The turnover of CD4(+)CD45RA(+) naive T cells was increased 2-fold, whereas that of CD4(-)CD45RA(+) naive T cells was marginally increased. B cells and NK cells also had increased turnover in SIV-infected macaques, averaging 3 and 2.5% per day, respectively. For all cell types studied here the daily turnover rate increased with the decrease of the CD4 count that accompanied SIV infection. As a consequence, the turnover rates of CD4(+) T cells, CD4(-) T cells, B cells, and NK cells within each monkey are strongly correlated. This suggests that the cellular turnover of different lymphocyte populations is governed by a similar process which one could summarize as "generalized immune activation." Because the viral load and the CD4 T cell count are negatively correlated we cannot determine which of the two plays the most important role in this generalized immune activation.     

Recognition and killing of human and murine pancreatic beta cells by the NK receptor NKp46

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by β cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human β cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse β cell ligands. We show that human β cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the NKp46 ligand is detected on human β cells already at the embryonic stage and that it appears on murine β cells only following birth. Because the NKp46 ligand is detected on healthy β cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with β cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its β cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr(125) and Asn(216), are critical for this recognition.     

Mobilization of NK cells by exercise: downmodulation of adhesion molecules on NK cells by catecholamines

The change of plasma catecholamine concentration correlates with the change of natural killer (NK) activity and NK cell number in peripheral blood mononuclear cells (PBMC) during and after moderate exercise. We studied the causal relation between exercise-induced catecholamine and expression of adhesion molecules on NK cells during and after exercise. The expression of CD44 and CD18 on CD3(-)CD56(+) NK cells was significantly reduced during exercise (P < 0.01). When PBMC were stimulated with 10(-8)M norepinephrine in vitro, the expression of these adhesion molecules on CD3(-)CD56(+) NK cells was downmodulated within 30 min. The binding capacity of NK cells to a CD44 ligand, hyaluronate, was reduced by the stimulation with norepinephrine (P < 0.01). The intravenous injection of norepinephrine in mice decreased the expression of CD44 and CD18 on CD3(-)NK1.1(+) cells (P < 0.01) and increased the number of CD3(-)NK1.1(+) cells in PBMC (P < 0.01). These findings suggest that exercise-induced catecholamines modulate the expression of adhesion molecules on NK cells, resulting in the mobilization of NK cells into the circulation.     

Emergence of CD8+ T cells expressing NK cell receptors in influenza A virus-infected mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8(+) T cell population at day 10 postinfection. Almost all of the CD8(+)NK1.1(+) cells were CD8alphabeta(+)CD3(+)TCRalphabeta(+) and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-gamma after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1(-/-) mice. These results demonstrate that a significant proportion of CD8(+) T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8(+) T cell effector functions during viral infection.     

Targeting of human dendritic cells by autologous NK cells

NK cells have the capacity to spontaneously kill tumor cell lines, in particular cell lines of hemopoietic origin. In contrast, they do not generally kill nontransformed autologous cells. However, here we demonstrate that short-term activated polyclonal human NK cells, as well as human NK cell lines, efficiently lyse autologous dendritic cells (DC) derived from peripheral blood monocytes as well as Langerhans-like cells derived from CD34+ stem cells isolated from umbilical cord blood. Lysis of autologous DC by short-term activated NK cells and NK cell lines was dependent on granule exocytosis, since total abrogation of lysis was observed in the presence of EGTA. Induction of DC maturation by LPS, monocyte conditioned media (MCM), or stimulation through CD40 ligand (CD40L) rendered the DC less susceptible to lysis by NK cells. Infection of DC with influenza virus was likewise associated with a reduced susceptibility to lysis by NK cells. Thus, susceptibility to lysis by autologous NK cells is a particular property of immature DC. The present results are discussed in relation to the ability of DC to interact with NK cells and to the ability of NK cells to regulate development of specific immunity.     

Fractalkine-mediated endothelial cell injury by NK cells

Endothelial cells (ECs) are primary targets of immunological attack, and their injury can lead to vasculopathy and organ dysfunction in vascular leak syndrome and in rejection of allografts or xenografts. A newly identified CX3C-chemokine, fractalkine, expressed on activated ECs plays an important role in leukocyte adhesion and migration. In this study we examined the functional roles of fractalkine on NK cell activity and NK cell-mediated endothelial cell injury. Freshly separated NK cells expressed the fractalkine receptor (CX3CR1) determined by FACS analysis and efficiently adhered to immobilized full-length fractalkine, but not to the truncated forms of the chemokine domain or mucin domain, suggesting that fractalkine functions as an adhesion molecule on the interaction between NK cells and ECs. Soluble fractalkine enhanced NK cell cytolytic activity against K562 target cells in a dose- and time-dependent manner. This enhancement correlated well with increased granular exocytosis from NK cells, which was completely inhibited by the G protein inhibitor, pertussis toxin. Transfection of fractalkine cDNA into ECV304 cells or HUVECs resulted in increased adhesion of NK cells and susceptibility to NK cell-mediated cytolysis compared with control transfection. Moreover, both enhanced adhesion and susceptibility of fractalkine-transfected cells were markedly suppressed by soluble fractalkine or anti-CX3CR1 Ab. Our results suggest that fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage, which may result in vascular injury.     

Methionine dependence of virus-infected cells

Mouse embryo cells nonproductively infected with human cytomegalovirus differed from noninfected cells by the impaired ability to grow in the medium containing homocysteine instead of methionine. Virus infection of mouse embryo cells grown in both kinds of media resulted in the increase of protein synthesis. In the infected cells grown on homocysteine this increase was followed by a quick decrease. The effects of homocysteine substitution could be abolished by the addition of low amounts of methionine (0.1 mM). Methionine uptake in the infected cells grown on homocysteine for 48 h was significantly higher than that in the noninfected cells.     

Interleukin-15 increases effector memory CD8+ t cells and NK Cells in simian immunodeficiency virus-infected macaques

Interleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+ T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+ T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 microg/kg of body weight, n = 3; high dose of IL-15, 100 microg/kg, n = 3; control [saline], n = 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3- NK cells. Furthermore, CD8+ T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA-CD62L- and CD45RA+CD62L- effector memory CD8+ T cells. Expression of Ki-67 in the CD8+ T cells indicated expansion of CD8+ T cells and not redistribution. IL-15 did not affect CD4+ T-cell, B-cell, and CD14+ macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+ T cells and NK cells by IL-15 further supports IL-15's possible therapeutic use in viral infections such as HIV infection.     

Polarization of allogeneic T-cell responses by influenza virus-infected dendritic cells

The developing immune response in the lymph nodes of mice infected with influenza virus has both Th1- and Th2-type characteristics. Modulation of the interactions between antigen-presenting cells and T cells is one mechanism that may alter the quality of the immune response. We have previously shown that the ability of dendritic cells (DC) to stimulate the proliferation of alloreactive T cells is changed by influenza virus due to viral neuraminidase (NA) activity. Here we show that DC infected with influenza virus A/PR/8/34 (PR8) stimulate T cells to produce different types of cytokines in a dose-dependent manner. Optimal amounts of the Th1-type cytokines interleukin-2 (IL-2) and gamma interferon (IFN-gamma) were produced from T cells stimulated by DC infected with low doses of PR8, while the Th2-type cytokines IL-4 and IL-10 were produced only in response to DC infected with high doses of PR8. IL-2 and IFN-gamma levels corresponded with T-cell proliferation and were dependent on the activity of viral NA on the DC surface. In contrast, IL-4 secretion required the treatment of T cells with NA. Since viral particles were released only from DC that are infected with high doses of PR8, our results suggest that viral NA on newly formed virus particles desialylates T-cell surface molecules to facilitate a Th2-type response. These results suggest that the activity of NA may contribute to the mixed Th-type response observed during influenza virus infection.     

Killer cells of feline leukemia virus- and feline sarcoma virus-infected transformed cells: the role of NK, ADCC, and in vitro generated cytotoxic cells

Feline white blood cells (WBC) manifested a primary in vitro mixed lymphocyte tumor cell culture (MLTC) proliferative response to feline leukemia virus-feline sarcoma virus (FeLV-FeSV)-infected transformed target cells, which reached a peak at Day 15. Furthermore, primary in vitro MLTC cultures generated cytotoxic killer cells capable of killing a variety of targets in non-major histocompatibility gene complex restricted fashion, and effector cells were capable of killing targets introduced repeatedly into cultures over a 49-day period. The presence of feline fibrosarcoma (f-sarc) stimulators was the primary driving force for proliferation and generation of killing because exogenous IL-2 conditioned medium did not appreciably increase the yield of killer cells generated in vitro. WBC cultured without f-sarc stimulators with or without IL-2 supplementation also generated killer (K) cells but at a low level. The killer cell population was composed of approximately 50% lymphocytes and 50% monocytes. Cats had K cells functional in antibody-dependent cell-mediated cytotoxicity against FeLV-coated chicken red blood cells but not against any FeLV-FeSV-infected transformed targets tested. Natural killer (NK) cell activity to any targets tested was not found. Although no evidence was found for K or NK cell activity against FeLV-FeSV-infected transformed cells, feline WBC were readily able to generate killer cells in vitro and it is probable that this cell-mediated immune potential is functionally important in vivo.