The CD85j+ NK cell subset potently controls HIV-1 replication in autologous dendritic cells

Natural killer (NK) cells and dendritic cells (DC) are thought to play critical roles in the first phases of HIV infection. In this study, we examined changes in the NK cell repertoire and functions occurring in response to early interaction with HIV-infected DC, using an autologous in vitro NK/DC coculture system. We show that NK cell interaction with HIV-1-infected autologous monocyte-derived DC (MDDC) modulates NK receptor expression. In particular, expression of the CD85j receptor on NK cells was strongly down-regulated upon coculture with HIV-1-infected MDDC. We demonstrate that CD85j(+) NK cells exert potent control of HIV-1 replication in single-round and productively HIV-1-infected MDDC, whereas CD85j(-) NK cells induce a modest and transient decrease of HIV-1 replication. HIV-1 suppression in MDCC by CD85j(+) NK cells required cell-to-cell contact and did not appear mediated by cytotoxicity or by soluble factors. HIV-1 inhibition was abolished when NK-MDDC interaction through the CD85j receptor was blocked with a recombinant CD85j molecule, whereas inhibition was only slightly counteracted by blocking HLA class I molecules, which are known CD85j ligands. After masking HLA class I molecules with specific antibodies, a fraction of HIV-1 infected MDDC was still strongly stained by a recombinant CD85j protein. These results suggest that CD85j(+) NK cell inhibition of HIV-1 replication in MDDC is mainly mediated by CD85j interaction with an unknown ligand (distinct from HLA class I molecules) preferentially expressed on HIV-1-infected MDDC.     

Human immunodeficiency virus type 1 infection is associated with increased NK cell polyfunctionality and higher levels of KIR3DL1+ NK cells in ugandans carrying the HLA-B Bw4 motif

Natural killer (NK) cells are important innate effector cells controlled by an array of activating and inhibitory receptors. Some alleles of the inhibitory killer-cell immunoglobulin-like receptor KIR3DL1 in combination with its HLA class I ligand Bw4 have been genetically associated with slower HIV-1 disease progression. Here, we observed that the presence of HLA-B Bw4 was associated with elevated frequencies of KIR3DL1(+) CD56(dim) NK cells in chronically HIV-1-infected individuals from the rural district of Kayunga, Uganda. In contrast, levels of KIR2DL1(+) CD56(dim) NK cells were decreased, and levels of KIR2DL3(+) CD56(dim) NK cells were unchanged in infected subjects carrying their respective HLA-C ligands. Furthermore, the size of the KIR3DL1(+) NK cell subset correlated directly with viral load, and this effect occurred only in HLA-B Bw4(+) patients, suggesting that these cells expand in response to viral replication but may have relatively poor antiviral capacity. In contrast, no association with viral load was present for KIR2DL1(+) and KIR2DL3(+) NK cells. Interestingly, chronic HIV-1 infection was associated with an increased polyfunctional response in the NK cell compartment, and, upon further investigation, KIR3DL1(+) CD56(dim) NK cells exhibited a significantly increased functional response in the patients carrying HLA-B Bw4. These results indicate that chronic HIV-1 infection is associated with increased NK cell polyfunctionality and elevated levels of KIR3DL1(+) NK cells in Ugandans carrying the HLA-B Bw4 motif.     

Increased natural killer cell activity in viremic HIV-1 infection

NK cells are a subset of granular lymphocytes that are critical in the innate immune response to infection. These cells are capable of killing infected cells and secreting integral cytokines and chemokines. The role that this subset of cytolytic cells plays in HIV infection is not well understood. In this study, we dissected the function of NK cells in viremic and aviremic HIV-1-infected subjects, as well as HIV-1-negative control individuals. Despite reduced NK cell numbers in subjects with ongoing viral replication, these cells were significantly more active in secreting both IFN-gamma and TNF-alpha than NK cells from aviremic subjects or HIV-1-negative controls. In addition, NK cells in subjects with detectable viral loads expressed significantly higher levels of CD107a, a marker of lysosomal granule exocytosis. The expression of CD107a correlated with NK cell-mediated cytokine secretion and cytolytic activity as well as with the level of viral replication, suggesting that CD107a represents a good marker for the functional activity of NK cells. Finally, killer Ig-related receptor+ NK cells were stable or elevated in viremic subjects, while the numbers of CD3-/CD56+/CD94+ and CD3-/CD56+/CD161+ NK cells were reduced. Taken together, these data demonstrate that viremic HIV-1 infection is associated with a reduction in NK cell numbers and a perturbation of NK cell subsets, but increased overall NK cell activity.     

NK cell function in HIV-1 infection

NK cells are critical effector cells of the innate immune response to malignancy and infection. These cells have a wide array of direct antiviral activities and have been critically implicated in the regulation and induction of an effective adaptive immune response. Although the pivotal role of this cell subset in the context of a number of viral infections is well established, the role of NK cells in HIV-1 infection is less well understood. Recent data has demonstrated the association between an NK cell receptor, KIR3DS1, and it's ligand, HLA-Bw4 with an isoleucine at position 80, and slower disease progression. This data suggests that NK cells may play an essential role in the control of HIV-1 disease, and has provided the impetus to begin to better understand the role of this cell subset in the context of HIV-1 infection, replication, and pathogenesis. Here we present a review of the literature pertaining to both the effect of HIV-1 infection on NK cell activity and the potential role that this subset of cells may play in controlling HIV-1 disease.     

Antibody-mediated neutralization of primary human immunodeficiency virus type 1 isolates: investigation of the mechanism of inhibition

Human immunodeficiency virus type 1 (HIV-1) neutralization occurs when specific antibodies, mainly those directed against the envelope glycoproteins, inhibit infection, most frequently by preventing the entry of the virus into target cells. However, the precise mechanisms of neutralization remain unclear. Previous studies, mostly with cell lines, have produced conflicting results involving either the inhibition of virus attachment or interference with postbinding events. In this study, we investigated the mechanisms of neutralization by immune sera and compared the inhibition of peripheral blood mononuclear cells (PBMC) infection by HIV-1 primary isolates (PI) with the inhibition of T-cell line infection by T-cell line-adapted (TCLA) strains. We followed the kinetics of neutralization to determine at which step of the viral cycle the antibodies act. We found that neutralization of the TCLA strain HIV-1MN/MT-4 required an interaction between antibodies and cell-free virions before the addition of MT-4 cells, whereas PI were neutralized even after adsorption onto PBMC. In addition, the dose-dependent inhibition of HIV-1MN binding to MT-4 cells was strongly correlated with serum-induced neutralization. In contrast, neutralizing sera did not reduce the adhesion of PI to PBMC. Postbinding inhibition was also detected for HIV-1MN produced by and infecting PBMC, demonstrating that the mechanism of neutralization depends on the target cell used in the assay. Finally, we considered whether the different mechanisms of neutralization may reflect the recognition of qualitatively different epitopes on the surface of PI and HIV-1MN or whether they reflect differences in virus attachment to PBMC and MT-4 cells.     

DCs and NK cells: critical effectors in the immune response to HIV-1

Dendritic cells (DCs) and natural killer (NK) cells have central roles in antiviral immunity by shaping the quality of the adaptive immune response to viruses and by mediating direct antiviral activity. HIV-1 infection is characterized by a severe dysregulation of the antiviral immune response that starts during early infection. This Review describes recent insights into how HIV-1 infection affects DC and NK cell function, and the roles of these innate immune cells in HIV-1 pathogenesis. The importance of understanding DC and NK cell crosstalk during HIV infection for the development of effective antiviral strategies is also discussed.     

Evasion from NK cell-mediated immune responses by HIV-1

Human immunodeficiency virus type 1 (HIV-1) mostly owes its success to its ability to evade host immune responses. Understanding viral immune escape mechanisms is a prerequisite to improve future HIV-1 vaccine design. This review focuses on the strategies that HIV-1 has evolved to evade recognition by natural killer (NK) cells.     

Demonstration of the antiviral role of natural killer cells in vivo with a natural killer cell-specific monoclonal antibody (NK 1.1)

A monoclonal antibody (NK 1.1) to mouse natural killer (NK) cells selectively depleted NK cell activity in virus-infected mice without significantly depressing other immune functions, including the development of virus-specific cytotoxic T cells. NK cell depletion with this antibody resulted in markedly enhanced plaque-forming unit titers of some (murine cytomegalo, Pichinde) but not other (mouse hepatitis, lymphocytic choriomeningitis) viruses. This confirms that NK cells do play a role in regulating certain infections and shows that this antibody provides a convenient tool for examining the role of NK cells in viral infections.     

In vitro inhibition of HIV-1 replication in autologous CD4~+ T cells indicates viral containment by multifactorial mechanisms

HIV-1-specific cytotoxic T lymphocytes(CTLs) and neutralizing antibodies(NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells,primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro,which was mediated by natural killer cells(NKs) and dependent on an Fc-Fc receptor interaction.Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4~+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.     

Natural killer cells in HIV-1 infection: role of NK cell-mediated non-cytolytic mechanisms in pathogenesis of HIV-1 infection

Natural killer (NK) cells exhibit both cytolytic and non-cytolytic effector functions against HIV-infected targets. Their precise role in immunopathogenesis of HIV-1 infection is yet to be fully understood. This review addresses the non-cytolytic functions exhibited by NK cells, their potential role in pathogenesis of HIV-1 infection and the effect of HIV-1 viremia on NK cell functions. Activated NK cells are capable of secreting CC-chemokines and suppressing HIV-1 replication in a non-cytolytic fashion. However, HIV-1 viremia suppresses the ability of NK cells to secrete CC-chemokines. Suppression of HIV-1 viremia by highly active antiretroviral therapy (HAART) restores the ability of NK cells to secrete CC-chemokines and suppress endogenous HIV-1 replication by non-cytolytic mechanisms. Better understanding of the mechanisms involved in HIV-1-NK cell interactions would be helpful in delineating novel therapeutic strategics against HIV-1.     

Natural killer cell function is well preserved in asymptomatic human immunodeficiency virus type 2 (HIV-2) infection but similar to that of HIV-1 infection when CD4 T-cell counts fall

Natural killer (NK) cells are potent effectors of natural immunity and their activity prevents human immunodeficiency virus type 1 (HIV-1) viral entry and viral replication. We sought to determine whether NK immune responses are associated with different clinical course of HIV-1 and HIV-2 infections. A cross-sectional analysis of NK cell responses was undertaken in 30 HIV-1 and 30 HIV-2 subjects in each of three categories of CD4(+)-T-cell counts (>500, 200 to 500, and <200 cells/microl) and in 50 HIV-uninfected control subjects. Lytic activity and gamma interferon (IFN-gamma) secretion were measured by chromium release and enzyme-linked immunospot assays, respectively. Flow cytometry was used to assess intracellular cytokines and chemokines. Levels of NK cytotoxicity were significantly higher in HIV-2 than in HIV-1 infections in subjects with high CD4(+)-T-cell counts and were similar to that of the healthy controls. In these HIV-2 subjects, cytolytic activity was positively correlated to NK cell count and inversely related to plasma viremia. Levels of intracellular MIP-1beta, RANTES, tumor necrosis factor alpha, and IFN-gamma produced by NK CD56(bright) cells were significantly higher in HIV-2- than HIV-1-infected subjects with high CD4(+)-T-cell counts but fell to similar levels as CD4 counts dropped. The data suggest efficient cytolytic and chemokine-suppressive activity of NK cells early in HIV-2 infection, which is associated with high CD4(+) T-cell counts. Enhancement of these functions may be important in immune-based therapy to control HIV disease.     

NK cells and apoptosis

Natural killer (NK) cells are a cell of the innate immune system that play an important role in the early response to viral infections and tumours. Natural killer cells are cytolytic, and secrete cytokines that influence the developing antigen-specific immune response. In the present article the NK cell surface molecules regulating effector function, the NK cell effector mechanisms involved in apoptosis, and the role of NK cell effector mechanisms in immune responses are reviewed.     

Involvement of Fc gamma RI (CD64) in the mechanism of HIV-1 inhibition by polyclonal IgG purified from infected patients in cultured monocyte-derived macrophages

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcgammaRs in HIV-1 inhibition, F(ab')(2) and Fab of these Igs were produced. Results indicated that both F(ab')(2) and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcgammaRs Ab strengthen the participation of the FcgammaRs, and in particular of FcgammaRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.     

Increased neutralization sensitivity and reduced replicative capacity of human immunodeficiency virus type 1 after short-term in vivo or in vitro passage through chimpanzees

Development of disease is extremely rare in chimpanzees when inoculated with either T-cell-line-adapted neutralization-sensitive or primary human immunodeficiency virus type 1 (HIV-1), at first excluding a role for HIV-1 neutralization sensitivity in the clinical course of infection. Interestingly, we observed that short-term in vivo and in vitro passage of primary HIV-1 isolates through chimpanzee peripheral blood mononuclear cells (PBMC) resulted in a neutralization-sensitive phenotype. Furthermore, an HIV-1 variant reisolated from a chimpanzee 10 years after experimental infection was still sensitive to neutralization by soluble CD4, the CD4 binding site recognizing antibody IgG1b12 and autologous chimpanzee serum samples, but had become relatively resistant to neutralization by polyclonal human sera and neutralizing monoclonal antibodies. The initial adaptation of HIV-1 to replicate in chimpanzee PBMC seemed to coincide with a selection for viruses with low replicative kinetics. Neither coreceptor usage nor the expression level of CD4, CCR5, or CXCR4 on chimpanzee PBMC compared to human cells could explain the phenotypic changes observed in these chimpanzee-passaged viruses. Our data suggest that the increased neutralization sensitivity of HIV-1 after replication in chimpanzee cells may in part contribute to the long-term asymptomatic HIV-1 infection in experimentally infected chimpanzees.     

Single-stranded RNA derived from HIV-1 serves as a potent activator of NK cells

Persistent immune activation is a hallmark of chronic viremic HIV-1 infection. Activation of cells of the innate immune system, such as NK cells, occurs rapidly upon infection, and is sustained throughout the course of the disease. However, the precise underlying mechanism accounting for the persistent HIV-1-induced activation of NK cells is poorly understood. In this study, we assessed the role of uridine-rich ssRNA derived from the HIV-1 long terminal repeat (ssRNA40) on activation of NK cells via TLR7/8. Although dramatic activation of NK cells was observed following stimulation of PBMC with ssRNA40, negligible activation was observed following stimulation of purified NK cells despite their expression of TLR8 mRNA and protein. The functional activation of NK cells by this HIV-1-encoded TLR7/8 ligand could not be reconstituted with exogenous IL-12, IFN-alpha, or TNF-alpha, but was critically dependent on the direct contact of NK cells with plasmacytoid dendritic cells or CD14(+) monocytes, indicating an important level of NK cell cross-talk and regulation by accessory cells during TLR-mediated activation. Coincubation of monocyte/plasmacytoid dendritic cells, NK cells, and ssRNA40 potentiated NK cell IFN-gamma secretion in response to MHC-devoid target cells. Studies using NK cells derived from individuals with chronic HIV-1 infection demonstrated a reduction of NK cell responsiveness following stimulation with TLR ligands in viremic HIV-1 infection. These data demonstrate that HIV-1-derived TLR ligands can contribute to the immune activation of NK cells and may play an important role in HIV-1-associated immunopathogenesis and NK cell dysfunction observed during acute and chronic viremic HIV-1 infection.     

The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR

The human immunodeficiency virus type 1 (HIV-1) evades the immune responses of natural killer (NK) cells through mechanisms that have been partially deciphered. Here we show that in HIV-1-infected T lymphocytes, the early viral Nef protein downmodulates PVR (CD155, Necl-5), a ligand for the activating receptor DNAM-1 (CD226) expressed by all NK cells, CD8(+) T cells, and other cell types. This novel Nef activity is conserved by Nef proteins of laboratory HIV-1 strains (NL4-3, SF2) and of a patient-derived virus, but it is not maintained by HIV-2. Nef uses the same motifs to downregulate PVR and HLA-I molecules, likely by the same mechanisms. Indeed, as previously demonstrated for HLA-I, Nef reduces the total amounts of cell-associated PVR. Optimal downregulation of cell surface PVR by Nef also requires the presence of the late viral factor Vpu. In line with PVR reduction, the NK cell-mediated lysis of T cells infected by a wild-type but not Nef-deficient virus is virtually abrogated upon blocking of both DNAM-1 and another activating receptor, NKG2D, previously shown to mediate killing of HIV-infected cells. Together, these data demonstrate that the PVR downmodulation by Nef and Vpu is a strategy evolved by HIV-1 to prevent NK cell-mediated lysis of infected cells. The PVR downregulation reported here has the potential to affect the immune responses of other DNAM-1-positive cells besides NK cells and to alter multiple PVR-mediated cellular processes, such as adhesion and migration, and may thus greatly influence HIV-1 pathogenesis.     

Innate immune response of the human host to exposure with herpes simplex virus type 1: in vitro control of the virus infection by enhanced natural killer activity via interleukin-15 induction

Infections with herpes simplex virus type 1 (HSV-1) in humans and in animal models are accompanied by enhanced natural killer (NK) activity. In vitro, HSV-1 also enhances the NK activity of human peripheral blood mononuclear cells (PBMC). The molecular basis of this enhanced NK activity, however, is not well characterized. We investigated the role of human interleukin-15 (IL-15) in this phenomenon and report here that HSV-1-mediated enhanced NK activity was abrogated by neutralizing antibodies for IL-15 but not for other cytokines (i.e., IL-2, IL-12, gamma interferon [IFN-gamma], tumor necrosis factor alpha, or IFN-alpha). Anti-CD122 antibodies which block signaling through IL-2 receptor beta chain, and therefore neutralize the effects of IL-15 (and IL-2), also abrogated this enhancement. Furthermore, HSV-1 increased the levels of IL-15 mRNA and the production of IL-15 in HSV-1-infected PBMC cultures. The neutralization of IL-15 in cocultures of PBMC with HSV-1-infected cells significantly increased HSV-1 production. These results strongly suggest a role for IL-15 in the HSV-1-mediated in vitro enhancement of NK activity and in the PBMC-mediated suppression of HSV-1 replication.     

Regulation of NK cell activity by prostaglandin E2: the role of T cells

The influence of T cells on the production of prostaglandins (PGE2) and on PGE2-mediated regulation of natural killer (NK) activity was studied. Supernatants from peripheral blood mononuclear cells (PBMC) and from PBMC depleted of T cells ((PBMC)-T), both of which had been incubated in plastic petri dishes overnight, contained similar amounts of PGE2, as detected by radioimmunoassay and by their potential to inhibit NK activity of peripheral blood mononuclear cells in a 51Cr release assay with K 562 cells as the target population. However, the NK activity of PBMC was inhibited significantly more strongly (P less than 0.005) by PGE2-containing supernatants than was the NK activity of (PBMC)-T. In further assays, in which synthetic PGE2 in concentrations of 10(-4) and 10(-5)M was added, a significant inhibition of NK activity was observed in PBMC populations (P less than 0.05), but not in (PBMC)-T. Thus, T cells did not seem to be involved in the control of PGE2 production, but their presence was necessary for PGE2-mediated inhibition of NK activity.     

Interactions between natural killer cells and antibody Fc result in enhanced antibody neutralization of human immunodeficiency virus type 1

Antibodies can prevent lentivirus infections in animals and may play a role in controlling viral burden in established infection. In preventing and particularly in controlling infection, antibodies likely function in the presence of large quantities of virus. In this study, we explored the mechanisms by which antibodies neutralize large inocula of human immunodeficiency virus type 1 (HIV-1) on different target cells. Immunoglobulin G (IgG) from HIV-infected patients was tested for neutralizing activity against primary R5 strains of HIV-1 at inocula ranging from 100 to 20,000 50% tissue culture infective doses. At all virus inocula, inhibition by antibody was enhanced when target cells for virus growth were monocyte-depleted, peripheral blood mononuclear cells (PBMCs) rather than CD4(+) lymphocytes. However, enhanced inhibition on PBMCs was greatest with larger amounts of virus. Depleting PBMCs of natural killer (NK) cells, which express Fc receptors for IgG (FcgammaRs), abrogated the enhanced antibody inhibition, whereas adding NK cells to CD4(+) lymphocytes restored inhibition. There was no enhanced inhibition on PBMCs when F(ab')(2) was used. Further experiments demonstrated that the release of beta-chemokines, most likely through FcgammaR triggering of NK cells, contributed modestly to the antiviral activity of antibody on PBMCs and that antibody-coated virus adsorbed to uninfected cells provided a target for NK cell-mediated inhibition of HIV-1. These results indicate that Fc-FcgammaR interactions enhance the ability of antibody to neutralize HIV-1. Since FcgammaR-bearing cells are always present in vivo, FcgammaR-mediated antibody function may play a role in the ability of antibody to control lentivirus infection.