HIV-infected CD4 Research Articles

Higher Expression of Activating Receptors on Cytotoxic NK Cells is Associated with Early Control on HIV-1C Multiplication.

Natural killer (NK) cells may be important in modulating HIV replication in early course of HIV infection. The effector function of NK cells is finely tuned by a balance between signals delivered by activating and inhibitory receptors. However, the influence of expression of these receptors on the early course of HIV replication and subsequent disease progression is not explored in the context of HIV-1C infection. The expression pattern of activating (NKp46, NKp44, NKp30, NKG2D, and NKG2C) and inhibitory (CD158b, NKG2A, and ILT2) receptors was determined in 20 patients with recent HIV-1C infection within 3–7 months of acquiring HIV infection and was compared with the expression pattern in individuals with progressive (N = 12), non-progressive HIV-1C infection (LTNPs, N = 12) and healthy seronegative individuals (N = 20). The association of the expression of these receptors on the rate of disease progression was assessed using viral load set point of recently infected individuals as a marker of disease progression. The study showed that higher cytotoxic potency of NK cells was associated with low viral load set point in recent HIV infection (r = −0.701; p = 0.0006) and higher CD4 counts (r = 0.720; p = 0.001). The expression of activating receptors (NKp46, NKp30, and NKG2D) on cytotoxic NK cells but not on regulatory NK cells was also significantly associated with low viral set point (p < 0.01) and viral load in LTNPs and progressors (p < 0.01). The study also indicated that cytotoxic NK cells might show the ability to specifically lyse HIV infected CD4 cells. This data collectively showed that early and sustained higher expression of activating receptors on cytotoxic NK cells could be responsible for increased cytotoxicity, reduced viral burden, and thus delaying the disease progression. The study to identify the molecular mechanism of the expression of these receptors in HIV infection will be helpful in further understanding of NK cell mediated control in early HIV infection.

HIV-1 NEF ligands predominate in the HLA class I of infected CD4+ T cells (APP2P.108)

Abstract Class I HLA reveal virus-derived ligands to cytotoxic T lymphocytes (CTL) whose function is to eliminate infected cells. The Los Alamos National Laboratory (LANL) database reports numerous HIV-1 CTL epitopes, none of which have elicited protective immunity in vaccine testing. The goal of this study was to determine the number and nature of HIV-1 ligands available for CTL targeting through presentation by the HLA class I of virus-infected cells. Class I presented peptides were recovered from immunoaffinity purified HLA-A*11:01 gathered from HIV-1 (NL4-3)-infected human CD4+ SUP-T1 T cells. These peptides were fractionated by RP-HPLC and mapped by tandem mass spectrometry (MS/MS). Seven HIV-derived peptides were confirmed by MS/MS as unique to infected cells. Twelve LANL HIV epitopes, including the well-studied Gag p24 epitope ACQGVGGPGHK (AK11), were clearly absent from the HLA-A*11:01 of infected cells. Of the 7 HIV-1 ligands found to be presented by A*11:01, 4 were derived from Nef, 2 from Gag, and 1 from Pol. When these 7 ligands were tested for T cell reactivity in a gamma interferon ELISPOT assay using PBMC from HIV-1 infected individuals (including elite controllers), the Nef-derived peptides were found to be highly reactive. These data demonstrate that the class I HLA of HIV-1 infected CD4+ T cells present a handful of viral peptide ligands for recognition by CTL and that reported HIV-1 CTL epitopes might not be presented by the HLA class I of infected cells.

HIV infection of CD4+ TH17 cells in the human female reproductive tract

HIV infection of CD4+ TH17 cells in the human female reproductive tract

High Avidity CD8+ T Cells Efficiently Eliminate Motile HIV-Infected Targets and Execute a Locally Focused Program of Anti-Viral Function

The dissemination of HIV from an initial site of infection is facilitated by motile HIV-infected CD4+ T-cells. However, the impact of infected target cell migration on antigen recognition by HIV-specific CD8+ T-cells is unclear. Using a 3D in vitro model of tissue, we visualized dynamic interactions between HIV-infected or peptide-pulsed CD4+ T-cells and HIV-specific CD8+ T-cells. CTLs engaged motile HIV-infected targets, but ∼50% of targets broke contact and escaped. In contrast, immobilized target cells were readily killed, indicating target motility directly inhibits CD8+ T-cell function. Strong calcium signals occurred in CTLs killing a motile target but calcium signaling was weak or absent in CTLs which permitted target escape. Neutralization of adhesion receptors LFA-1 and CD58 inhibited CD8+ T-cell function within the 3D matrix, demonstrating that efficient motile target lysis as dependent on adhesive engagement of targets. Antigen sensitivity (a convolution of antigen density, TCR avidity and CD8 coreceptor binding) is also critical for target recognition. We modulated this parameter (known as functional avidity but referred to here as “avidity” for the sake of simplicity) by exploiting common HIV escape mutations and measured their impact on CTL function at the single-cell level. Targets pulsed with low avidity mutant antigens frequently escaped while CTLs killed targets bearing high avidity antigen with near-perfect efficiency. CTLs engaged, arrested, and killed an initial target bearing high avidity antigen within minutes, but serial killing was surprisingly rare. CD8 cells remained committed to their initial dead target for hours, accumulating TCR signals that sustained secretion of soluble antiviral factors. These data indicate that high-avidity CD8+ T-cells execute an antiviral program in the precise location where antigen has been sensed: CTL effector functions are spatiotemporally coordinated with an early lytic phase followed by a sustained stationary secretory phase to control local viral infection.

HIV Nef and Vpu protect HIV-infected CD4+ T cells from antibody-mediated cell lysis through down-modulation of CD4 and BST2

BackgroundHIV proteins Nef and Vpu down-modulate various host factors to evade immune defenses. Indeed, the CD4 receptor is down-regulated by Nef and Vpu, whereas virion-tethering BST2 is depleted by Vpu. Antibody-dependent cell-mediated cytotoxicity (ADCC) is increasingly recognized as a potentially powerful anti-HIV response. Given that epitopes which are specific for ADCC-competent anti-HIV antibodies are transitionally exposed upon CD4-mediated HIV entry, we investigated whether by depleting CD4 and BST2, HIV could negatively affect ADCC function.ResultsUsing anti-envelope (Env) Abs A32 and 2G12 to trigger ADCC activity, we find that interactions between CD4 and Env within infected cells expose ADCC-targeted epitopes on cell-surface Env molecules, marking infected T cells for lysis by immune cells. We also provide evidence to show that by cross-linking nascent virions at the plasma membrane, hence increasing cell-surface Env density, BST2 further enhances the efficiency of this antiviral process. The heightened susceptibility of T cells infected with a virus lacking Nef and Vpu to ADCC was recapitulated when plasmas from HIV-infected patients were used as an alternative source of Abs.ConclusionsOur data unveil a mechanism by which HIV Nef and Vpu function synergistically to protect infected cells from ADCC and promote viral persistence. These findings also renew the potential practical relevance of ADCC function in vivo.

The tyrosine kinase inhibitor Dasatinib blocks in-vitro HIV-1 production by primary CD4+ T cells from HIV-1 infected patients

HIV reservoirs persistence despite antiretroviral therapy (ART) might be related to persistent immune activation and residual HIV production, requiring further therapeutic strategies. We demonstrated that the tyrosine kinase inhibitor (TKI) Dasatinib, used for chronic myeloid leukaemia, significantly blocks in vitro HIV1 production by 3.4 logs in HIV1-infected primary CD4 T lymphocytes, by inhibiting cell activation and proliferation, without cell toxicity. This molecule deserves to be investigated further for HIV cure strategies to hinder persistent immune activation and residual viral production.

The Majority of CD4 + T-Cell Depletion during Acute Simian-Human Immunodeficiency Virus SHIV89.6P Infection Occurs in Uninfected Cells

Untreated human immunodeficiency virus (HIV) infection is characterized by depletion of CD4(+) T cells, ultimately leading to the impairment of host immune defenses and death. HIV-infected CD4(+) T cells die from direct virus-induced apoptosis and CD8 T-cell-mediated elimination, but a broader and more profound depletion occurs in uninfected CD4(+) T cells via multiple indirect effects of infection. We fit mathematical models to data from experiments that tested an HIV eradication strategy in which five macaques with a proportion of CD4(+) T cells resistant to simian-human immunodeficiency virus (SHIV) entry were challenged with SHIV89.6P, a highly pathogenic dual-tropic chimeric SIV-HIV viral strain that results in rapid loss of both SHIV-susceptible and SHIV-resistant CD4(+) T cells. Our results suggest that uninfected (bystander) cell death accounts for the majority of CD4(+) T-lymphocyte loss, with at least 60% and 99% of CD4(+) T cell death occurring in uninfected cells during acute and established infection, respectively. Mechanisms to limit the profound indirect killing effects associated with HIV infection may be associated with immune preservation and improved long-term survival. HIV infection induces a massive depletion of CD4(+) T cells, leading to profound immunodeficiency, opportunistic infections, and eventually death. While HIV induces apoptosis (programmed cell death) by directly entering and replicating in CD4(+) T cells, uninfected CD4(+) T cells also undergo apoptosis due to ongoing toxic inflammation in the region of infection. In this paper, we use mathematical models in conjunction with data from simian-human immunodeficiency virus SHIV89.6P infection in macaques (a model of HIV infection in humans) to estimate the percentage of cell death that occurs in uninfected cells during the initial period of infection. We reveal that the vast majority of cell death occurs in these cells, which are not infected. The "bystander effects" that lead to enormous reductions in the number of uninfected CD4(+) T cells may be a target for future interventions that aim to limit the extent of damage caused by HIV.

Susceptibility to CD8 T-Cell–Mediated Killing Influences the Reservoir of Latently HIV-1–Infected CD4 T Cells

HIV-1 establishes a lifelong infection in the human body, but host factors that influence viral persistence remain poorly understood. Cell-intrinsic characteristics of CD4 T cells, the main target cells for HIV-1, may affect the composition of the latent viral reservoir by altering the susceptibility to CD8 T-cell-mediated killing. We observed that susceptibilities of CD4 T cells to CD8 T-cell-mediated killing, as determined in direct ex vivo assays, were significantly higher in persons with natural control of HIV-1 (elite controllers) than in individuals effectively treated with antiretroviral therapy. These differences were most pronounced in naive and in terminally differentiated CD4 T cells and corresponded to a reduced viral reservoir size in elite controllers. Interestingly, the highest susceptibility to CD8 T-cell-mediated killing and lowest reservoirs of cell-associated HIV-1 DNA was consistently observed in elite controllers expressing the protective HLA class I allele B57. These data suggest that the functional responsiveness of host CD4 T cells to cytotoxic effects of HIV-1-specific CD8 T cells can contribute to shaping the structure and composition of the latently infected CD4 T-cell pool.

Modelling the Drugs Therapy for HIV Infection with Discrete-Time Delay

A discrete-time-delay differential mathematical model that described HIV infection of CD4+T cells with drugs therapy is analyzed. The stability of the two equilibria and the existence of Hopf bifurcation at the positive equilibrium are investigated. Using the normal form theory and center manifold argument, the explicit formulas which determine the stability, the direction, and the period of bifurcating periodic solutions are derived. Numerical simulations are carried out to explain the mathematical conclusions.

Choice of antiretroviral therapy differentially impacts survival of HIV-infected CD4 T cells.

BackgroundHIV eradication strategies are now being evaluated in vitro and in vivo. A cornerstone of such approaches is maximal suppression of viral replication with combination antiretroviral therapy (ART). Since many antiretroviral agents have off target effects, and different classes target different components of the viral life cycle, we questioned whether different classes of ART might differentially affect the survival and persistence of productively HIV-infected CD4 T cells.MethodsIn vitro infections of primary CD4 T cells using clinical isolates of HIV-1 that were either protease inhibitor susceptible (HIV PI-S), or resistant (HIV PI-R) were treated with nothing, lopinavir, efavirenz or raltegravir. Cell viability, apoptosis, and the proportion of surviving cells that were P24 positive was assessed by flow cytometry.ResultsIn HIV PI-S infected primary cultures, all three antiretroviral agents decreased viral replication, and reduced the total number of cells that were undergoing apoptosis (P < 0.01) similarly. Similarly, in the HIV PI-R infected cultures, both efavirenz and raltegravir reduced viral replication and reduced apoptosis compared to untreated control (P < 0.01), while lopinavir did not, suggesting that HIV replication drives T cell apoptosis, which was confirmed by association by linear regression (P < 0.0001) . However since HIV protease has been suggested to directly induce apoptosis of infected CD4 T cells, and HIV PI are intrinsically antiapoptotic, we evaluated apoptosis in productively infected (HIV P24+) cells. More HIV p24 positive cells were apoptotic in the Efavirenz or raltegravir treated cultures than the lopinavir treated cultures (P = 0.0008 for HIV PI-R and P = 0.06 for the HIV PI-S), indicating that drug class impacts survival of productively infected CD4 T cells.ConclusionsInhibiting HIV replication with a PI, NNRTI or INSTI reduces total HIV-induced T cell apoptosis. However, blocking HIV replication with PI but not with NNRTI or INSTI promotes survival of productively HIV-infected cells. Thus, selection of antiretroviral agents may impact the success of HIV eradication strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/2052-8426-2-1) contains supplementary material, which is available to authorized users.

Numerical Analysis for a Fractional Differential Time-Delay Model of HIV Infection of CD4+T-Cell Proliferation under Antiretroviral Therapy

We study a fractional differential model of HIV infection ofCD4+T-cell, in which theCD4+T-cell proliferation plays an important role in HIV infection under antiretroviral therapy. An appropriate method is given to ensure that both the equilibria are asymptotically stable forτ≥0. We calculate the basic reproduction numberR0, the IFEE0, two IPEsE1*andE2*, and so on, and judge the stability of the equilibrium. In addition, we describe the dynamic behaviors of the fractional HIV model by using the Adams-type predictor-corrector method algorithm. At last, we extend the model to incorporate the term which we consider the loss of virion and a bilinear term during attacking the target cells.

Hopf Bifurcation and Stability of Periodic Solutions for Delay Differential Model of HIV Infection of CD4+T-cells

This paper deals with stability and Hopf bifurcation analyses of a mathematical model of HIV infection ofCD4+T-cells. The model is based on a system of delay differential equations with logistic growth term and antiretroviral treatment with a discrete time delay, which plays a main role in changing the stability of each steady state. By fixing the time delay as a bifurcation parameter, we get a limit cycle bifurcation about the infected steady state. We study the effect of the time delay on the stability of the endemically infected equilibrium. We derive explicit formulae to determine the stability and direction of the limit cycles by using center manifold theory and normal form method. Numerical simulations are presented to illustrate the results.

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway

Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4(+) T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein.

Negative Elongation Factor (NELF) Coordinates RNA Polymerase II Pausing, Premature Termination, and Chromatin Remodeling to Regulate HIV Transcription

A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4(+) T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4(+) T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency.

Nevirapine Inhibits the Anti-HIV Activity of CD8+ Cells

Antiretroviral therapy (ART) significantly reduced the CD8 cell noncytotoxic anti-HIV response in 12 HIV-1-infected subjects (P < 0.0001). In separate experiments, CD8(+) cells from long-term survivors were cocultured with HIV-infected CD4(+) cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8(+) cells from 4 of the 14 LTSs was reduced with exposure to 10 μM of nevirapine (P < 0.05). The antiviral activity of CD8(+) cells from 2 LTSs was inhibited by 5 μM of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8(+) cells and thus could influence the effectiveness of antiretroviral therapy.

Vaccine-induced plasma IgA specific for the C1 region of the HIV-1 envelope blocks binding and effector function of IgG

Analysis of correlates of risk of infection in the RV144 HIV-1 vaccine efficacy trial demonstrated that plasma IgG against the HIV-1 envelope (Env) variable region 1 and 2 inversely correlated with risk, whereas HIV-1 Env-specific plasma IgA responses directly correlated with risk. In the secondary analysis, antibody-dependent cellular cytotoxicity (ADCC) was another inverse correlate of risk, but only in the presence of low plasma IgA Env-specific antibodies. Thus, we investigated the hypothesis that IgA could attenuate the protective effect of IgG responses through competition for the same Env binding sites. We report that Env-specific plasma IgA/IgG ratios are higher in infected than in uninfected vaccine recipients in RV144. Moreover, Env-specific IgA antibodies from RV144 vaccinees blocked the binding of ADCC-mediating mAb to HIV-1 Env glycoprotein 120 (gp120). An Env-specific monomeric IgA mAb isolated from an RV144 vaccinee also inhibited the ability of natural killer cells to kill HIV-1-infected CD4(+) T cells coated with RV144-induced IgG antibodies. We show that monomeric Env-specific IgA, as part of postvaccination polyclonal antibody response, may modulate vaccine-induced immunity by diminishing ADCC effector function.

Effect of cellular activation on the antigen processing machinery of primary CD4 T cells. (P5029)

Abstract The killing of HIV-infected CD4 T cells by specific CD8 T cells (CTL) requires the presentation of peptide-MHC-I complexes produced during the intracellular degradation of viral proteins by the proteasome and other peptidases, to their cognate T cell receptors. HIV infection induces general cellular activation, which renders cells more susceptible to infection. Whether the activation state of cells alters the expression and activity of the antigen processing machinery and the kinetics and nature of epitopes displayed by MHC-I is not known despite its potential role in altering the efficiency of recognition of infected cells by CTL. We compared the expression and peptidase activities of the antigen processing machinery of primary CD4 T cells upon activation with anti-CD3/CD28 beads or with phytohemagglutinin. In primary CD4 T cells from HIV seronegative individuals, the hydrolytic activities of the proteasome, the expression of several of its subunits and of the peptidase TPP2 significantly increased upon activation. The mass spectrometry analysis of in vitro degradation of 2 long epitope-containing HIV peptides in extracts from CD3/CD28-activated CD4 T cells showed production of smaller and less antigenic peptides than in the non-activated samples. Reduced presentation of peptides by activated cells might lead to reduced recognition and slower clearance of activated infected cells by CTL thus allowing more time for viral replication and spread.

HIV triggers interleukin 21-mediated induction of granzyme B-secreting B cells with T cell regulatory potential. (P1441)

Abstract Certain regulatory lymphocyte subpopulations like regulatory T cells can suppress T cell expansion by granzyme B (GrB) secretion. Recently, we found that B cells can also produce GrB and acquire regulatory potential in response to interleukin (IL-)21. Since HIV is associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Here, we show that infection of CD4+ T cells with HIV 1 (NL4-3) induces strong expression of IL-21 without upregulation of CD40 ligand. We demonstrate that such IL-21+CD40Llow T cells secrete IL-21, thereby inducing GrB in cocultured B cells rather than supporting plasma cell differentiation. In line with these results, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of freshly isolated B cells from HIV-infected patients, but not healthy control B cells, express GrB. Of note, coculture of HIV-infected CD4+ T cells with GrB+ B cells results in GrB transfer, and strongly suppresses both T cell proliferation and viral replication as indicated by significantly reduced p24 levels. The observed effects are enhanced by IL-21, and reduced by GrB inhibition. In summary, GrB induction in HIV-specific B cells may play a role in decelerating expansion of HIV-infected CD4+ T cells, while interfering with their terminal differentiation into plasma cells.

Estradiol Reduces Susceptibility of CD4+ T Cells and Macrophages to HIV-Infection

The magnitude of the HIV epidemic in women requires urgent efforts to find effective preventive methods. Even though sex hormones have been described to influence HIV infection in epidemiological studies and regulate different immune responses that may affect HIV infection, the direct role that female sex hormones play in altering the susceptibility of target cells to HIV-infection is largely unknown. Here we evaluated the direct effect of 17-β-estradiol (E2) and ethinyl estradiol (EE) in HIV-infection of CD4+ T-cells and macrophages. Purified CD4+ T-cells and monocyte-derived macrophages were generated in vitro from peripheral blood and infected with R5 and X4 viruses. Treatment of CD4+ T-cells and macrophages with E2 prior to viral challenge reduced their susceptibility to HIV infection in a dose-dependent manner. Addition of E2 2 h after viral challenge however did not result in reduced infection. In contrast, EE reduced infection in macrophages to a lesser extent than E2 and had no effect on CD4+ T-cell infection. Reduction of HIV-infection induced by E2 in CD4+ T-cells was not due to CCR5 down-regulation, but was an entry-mediated mechanism since infection with VSV-G pseudotyped HIV was not modified by E2. In macrophages, despite the lack of an effect of E2 on CCR5 expression, E2–treatment reduced viral entry 2 h after challenge and increased MIP-1β secretion. These results demonstrate the direct effect of E2 on susceptibility of HIV-target cells to infection and indicate that inhibition of target cell infection involves cell-entry related mechanisms.

Interleukin-27 is a potent inhibitor of cis HIV-1 replication in monocyte-derived dendritic cells via a type I interferon-independent pathway.

IL-27, a member of the IL-12 family of cytokines, plays an important and diverse role in the function of the immune system. Whilst generally recognized as an anti-inflammatory cytokine, in addition IL-27 has been found to have broad anti-viral effects. Recently, IL-27 has been shown to be a potent inhibitor of HIV-1 infection in CD4+ T cells and macrophages. The main objective of this study was to see whether IL-27 has a similar inhibitory effect on HIV-1 replication in dendritic cells (DCs). Monocytes were differentiated into immature DCs (iDCs) and mature DCs (mDCs) with standard techniques using a combination of GM-CSF, IL-4 and LPS. Following differentiation, iDCs were infected with HIV-1 and co-cultured in the presence or absence of IL-27. IL-27 treated DCs were shown to be highly potent inhibitors of cis HIV-1, particularly of CCR5 tropic strains. Of note, other IL-12 family members (IL-12, IL-23 and IL-35) had no effect on HIV-1 replication. Microarray studies of IL-27 treated DCs showed no up-regulation of Type I (IFN) gene expression. Neutralization of the Type-I IFN receptor had no impact on the HIV inhibition. Lastly, IL-27 mediated inhibition was shown to act post-viral entry and prior to completion of reverse transcription. These results show for the first time that IL-27 is a potent inhibitor of cis HIV-1 infection in DCs by a Type I IFN independent mechanism. IL-27 has previously been reported to inhibit HIV-1 replication in CD4+ T cells and macrophages, thus taken together, this cytokine is a potent anti-HIV agent against all major cell types targeted by the HIV-1 virus and may have a therapeutic role in the future.