Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.     

Blockade of virus infection by human CD4+ T cells via a cytokine relay network

CD4(+) T cells directly participate in bacterial clearance through secretion of proinflammatory cytokines. Although viral clearance relies heavily on CD8(+) T cell functions, we sought to determine whether human CD4(+) T cells could also directly influence viral clearance through cytokine secretion. We found that IFN-gamma and TNF-alpha, secreted by IL-12-polarized Th1 cells, displayed potent antiviral effects against a variety of viruses. IFN-gamma and TNF-alpha acted directly to inhibit hepatitis C virus replication in an in vitro replicon system, and neutralization of both cytokines was required to block the antiviral activity that was secreted by Th1 cells. IFN-gamma and TNF-alpha also exerted antiviral effects against vesicular stomatitis virus infection, but in this case, functional type I IFN receptor activity was required. Thus, in cases of vesicular stomatitis virus infection, the combination of IFN-gamma and TNF-alpha secreted by human Th1 cells acted indirectly through the IFN-alpha/beta receptor. These results highlight the importance of CD4(+) T cells in directly regulating antiviral responses through proinflammatory cytokines acting in both a direct and indirect manner.     

Endothelial cells promote human immunodeficiency virus replication in nondividing memory T cells via Nef-, Vpr-, and T-cell receptor-dependent activation of NFAT

Human endothelial cells (ECs) enhance human immunodeficiency virus (HIV) replication within CD4(+) memory T cells by 50,000-fold in a Nef-dependent manner. Here, we report that EC-mediated HIV type 1 replication is also dependent on an intact vpr gene. Moreover, we demonstrate that despite a requirement for engaging major histocompatibility complex (MHC) class II molecules and costimulators, EC-stimulated virus-producing cells (p24(high) T cells) do not proliferate, nor are they arrested in the cell cycle. Rather, they are minimally activated, sometimes expressing CD69 but not CD25, HLA-DR, VLA-1, or effector cytokines. Blocking antibodies to interleukin 2 (IL-2), IL-6, IL-7, or tumor necrosis factor do not inhibit viral replication. Cyclosporine effectively inhibits viral replication, as does disruption of the NFAT binding site in the viral long terminal repeat. Furthermore, in the presence of ECs, suboptimal T-cell receptor (TCR) stimulation with phytohemagglutinin L supports efficient viral replication, and suboptimal stimulation with toxic shock syndrome toxin 1 leads to viral replication selectively in the TCR-stimulated, Vbeta2-expressing T cells. Collectively, these data indicate that ECs provide signals that promote Nef- and Vpr-dependent HIV replication in memory T cells that have been minimally activated through their TCRs. Our studies suggest a mechanism for HIV replication in vivo within the reservoir of circulating memory CD4(+) T cells that persist despite antiretroviral therapy and further suggest that maintenance of immunological memory by MHC class II-expressing ECs via TCR signaling may contribute to HIV rebound following cessation of antiretroviral therapy.     

Human cytomegalovirus differentially controls B cell and T cell responses through effects on plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs), the main producers of type I IFN in response to viral infection, are essential in antiviral immunity. In this study, we assessed the effect of human CMV (HCMV) infection on PDC function and on downstream B and T cell responses in vitro. HCMV infection of human PDCs was nonpermissive, as immediate-early but not late viral Ags were detected. HCMV led to partial maturation of PDCs and up-regulated MHC class II and CD83 molecules but not the costimulatory molecules CD80 and CD86. Regardless of viral replication, PDCs secreted cytokines after contact with HCMV, including IFN-alpha secretion that was blocked by inhibitory CpG, suggesting an engagement of the TLR7 and/or TLR9 pathways. In the presence of B cell receptor stimulation, soluble factors produced by HCMV-matured PDCs triggered B cell activation and proliferation. Through PDC stimulation, HCMV prompted B cell activation, but only induced Ab production in the presence of T cells or T cell secreted IL-2. Conversely, HCMV hampered the allostimulatory ability of PDCs, leading to decreased proliferation of CD4(+) and CD8(+) T cells. These findings reveal a novel mechanism by which HCMV differentially controls humoral and cell-mediate immune responses through effects on PDCs.     

HIV-1-specific interleukin-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function

Functional defects in cytotoxic CD8(+) T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8(+) T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication without treatment. HIV-specific triggering of IL-21 by CD4(+) T cells was significantly enriched in these persons (P = 0.0007), while isolated loss of IL-21-secreting CD4(+) T cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21(+) CD4(+) T cells in acute infection resulted in lower viral set points (P = 0.002). Moreover, IL-21 production by CD4(+) T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8(+) T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8(+) T cells showed an enhanced ability to efficiently inhibit viral replication in vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21(+) CD4(+) T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance for vaccine design.     

Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th2 cytokines and productive infection in tissue macrophages during late-stage infection

HIV-1 is dual-tropic for CD4+ T lymphocytes and macrophages, but virus production in the macrophages becomes manifest only during late-stage infection, after CD4+ T cell functions are lost, and when opportunistic pathogens begin to flourish. In this study, the SHIV/macaque model of HIV pathogenesis was used to assess the role of cytokines in regulating virus replication in the two cell types. We injected complete Freund's adjuvant (CFA) intradermally into SHIV(KU)-infected macaques, and infused Schistosoma mansoni eggs into the liver and lungs of others. Tissues examined from these animals demonstrated that macrophages induced by CFA did not support viral replication while those induced by S. mansoni eggs had evidence of productive infection. RT-PCR analysis showed that both Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10) were present in the CFA lesions but only the Th2 cytokines were found in the S. mansoni lesions. Follow-up studies in macaque cell cultures showed that whereas IFN-gamma caused enhancement of virus replication in CD4+ T cells, it curtailed viral replication in infected macrophages. In contrast, IL-4 enhanced viral replication in infected macrophages. These studies strongly suggest that cytokines regulate the sequential phases of HIV replication in CD4 T cells and macrophages.     

Defective dendritic cell function in HIV-infected patients receiving effective highly active antiretroviral therapy: neutralization of IL-10 production and depletion of CD4+CD25+ T cells restore high levels of HIV-specific CD4+ T cell responses induced by dendritic cells generated in the presence of IFN-alpha

We previously demonstrated that GM-CSF/IFN-alpha combination allowed the differentiation of monocytes from HIV-infected patients into dendritic cells (DCs) exhibiting high CD8(+) T cell stimulating abilities. The present study was aimed at characterizing the ability of DCs generated in the presence of GM-CSF and IFN-alpha to induce CD4 T cell responses. DCs were generated from monocytes of HIV-infected patients in the presence of GM-CSF with either IFN-alpha (IFN-DCs) or IL-4 (IL-4-DCs) for 7 days. Eleven patients receiving highly active antiretroviral therapy and exhibiting CD4 cell counts above 400/mm(3) and plasma HIV-RNA <50 copies/ml for at least 1 year were included in the study. Both DC populations were found to be defective in inducing autologous (in response to tuberculin or HIV-p24) or allogeneic CD4 T cell proliferation. Neutralization of IL-10 during the differentiation of IFN-DCs, but not during the DC-T cell coculture, significantly increased their ability to stimulate autologous CD4 T cell proliferation in response to tuberculin and allogeneic CD4 T cell proliferation (4.1-fold and 3.0-fold increases, respectively, at the DC to T cell ratio of 1:10). Moreover, IL-10 neutralization and CD4(+)CD25(+) T cell depletion synergistically act to dramatically increase HIV-p24-specific CD4 T cell responses induced by IFN-DCs (31.7-fold increase) but not responses induced by IL-4-DCs. Taken together, our results indicate that IFN-DCs are more efficient than IL-4-DCs to stimulate CD4(+) T cell proliferation, further supporting their use for immune-based therapy in HIV infection.     

CD16+ monocyte-derived macrophages activate resting T cells for HIV infection by producing CCR3 and CCR4 ligands

The CD16(+) monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16(+) and CD16(-) Mo subsets to render resting CD4(+) T cells permissive for HIV replication. We demonstrate that CD16(+) Mo preferentially differentiate into macrophages (Mphi) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16(+), but not CD16(-), Mo-derived Mphi from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16(-) Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3(+) and CCR4(+) T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16(+) Mo/Mphi. Thus, expansion of CD16(+) Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

TLR7/8 triggering exerts opposing effects in acute versus latent HIV infection

TLRs trigger innate immunity by recognizing conserved motifs of microorganisms. Recently, ssRNAs from HIV and influenza virus were shown to trigger TLR7 and 8. Thus, we hypothesized that HIV ssRNA, by triggering TLR7/8, affects HIV pathogenesis. Indeed, HIV ssRNA rendered human lymphoid tissue of tonsillar origin or PBMC barely permissive to HIV replication. The synthetic compound R-848, which also triggers TLR7/8, showed similar anti-HIV activity. Loss of R-848's activity in lymphoid tissue depleted of B cells suggested a role for B cells in innate immunity. TLR7/8 triggering appears to exert antiviral effects through soluble factors: conditioned medium reduced HIV replication in indicator cells. Although a number of cytokines and chemokines were increased upon adding R-848 to lymphoid tissue, blocking those cytokines/chemokines (i.e., IFN-alpha receptor, IFN-gamma, MIP-1alpha, -1beta, RANTES, and stromal cell-derived factor-1) did not result in the reversal of R-848's anti-HIV activity. Thus, the nature of this soluble factor(s) remains unknown. Unlike lymphoid tissue acutely infected with HIV, triggering latently infected promonocytic cells induced the release of HIV virions. The anti-HIV effects of triggering TLR7/8 may inhibit rapid killing, while pro-HIV effects may guarantee a certain replication level. Compounds triggering TLR7/8 may be attractive drug candidates to purge latent HIV while preventing new infections.     

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.     

IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells

CD4(+) T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-alpha, but not TGF-beta, induces naive CD4(+) T cells derived from cord blood to differentiate into Tr1 cells: IL-10(+)IFN-gamma(+)IL-2(-/low)IL-4(-). Naive CD4(+) T cells derived from peripheral blood require both exogenous IL-10 and IFN-alpha for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-beta. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-alpha strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.     

Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells

Infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus produces acute and chronic demyelination. The contributions of perforin-mediated cytolysis and gamma interferon (IFN-gamma) secretion by CD8(+) T cells to the control of infection and the induction of demyelination were examined by adoptive transfer into infected SCID recipients. Untreated SCID mice exhibited uncontrolled virus replication in all CNS cell types but had little or no demyelination. Memory CD8(+) T cells from syngeneic wild-type (wt), perforin-deficient, or IFN-gamma-deficient (GKO) donors all trafficked into the infected CNS in the absence of CD4(+) T cells and localized to similar areas. Although CD8(+) T cells from all three donors suppressed virus replication in the CNS, GKO CD8(+) T cells expressed the least antiviral activity. A distinct viral antigen distribution in specific CNS cell types revealed different mechanisms of viral control. While wt CD8(+) T cells inhibited virus replication in all CNS cell types, cytolytic activity in the absence of IFN-gamma suppressed the infection of astrocytes, but not oligodendroglia. In contrast, cells that secreted IFN-gamma but lacked cytolytic activity inhibited replication in oligodendroglia, but not astrocytes. Demyelination was most severe following viral control by wt CD8(+) T cells but was independent of macrophage infiltration. These data demonstrate the effective control of virus replication by CD8(+) T cells in the absence of CD4(+) T cells and support the necessity for the expression of distinct effector mechanisms in the control of viral replication in distinct CNS glial cell types.     

Thymocyte-Thymic Epithelial Cell Interaction Leads to High-Level Replication of Human Immunodeficiency Virus Exclusively in Mature CD4+ CD8− CD3+ Thymocytes: a Critical Role for Tumor Necrosis Factor and Interleukin-7

This work aims at identifying the thymocyte subpopulation able to support human immunodeficiency virus (HIV) replication under the biological stimuli of the thymic microenvironment. In this report we demonstrate that interaction with thymic epithelial cells (TEC) induces a high-level replication of the T-tropic primary isolate HIV-1(B-LAIp) exclusively in the mature CD4(+) CD8(-) CD3(+) thymocytes. Tumor necrosis factor (TNF) and interleukin-7 (IL-7), secreted during this interaction, are critical cytokines for HIV long terminal repeat transactivation through NF-kappaB-dependent activation. TNF is the major inducer of NF-kappaB and particularly of the p50-p65 complex, whereas IL-7 acts as a cofactor by sustaining the expression of the p75 TNF receptor. The requirement for TNF is further confirmed by the observation that the inability of the intermediate CD4(+) CD8(-) CD3(-) thymocytes to replicate the virus is associated with a defect in TNF production during their interaction with TEC and correlates with the absence of nuclear NF-kappaB activity in these freshly isolated thymocytes. Addition of exogenous TNF to the intermediate thymocyte cultures induces NF-kappaB activity and is sufficient to promote HIV replication in the cocultures with TEC. The other major subpopulation expressing the CD4 receptor, namely, the double-positive (DP) CD4(+) CD8(+) CD3(+/-) thymocytes, despite the entry of the virus, do not produce a significant level of virus, presumably because they are unresponsive to TNF and IL-7. Together, these data suggest that in vivo, despite an efficient entry of the virus in all the CD4(+) subpopulations, a high viral load may be generated exclusively within the mature CD4(+) CD8(-) CD3(+) subset of thymocytes. However, under conditions of inflammatory response after infection, TNF might also be present in the intermediate thymocyte compartment, leading to efficient HIV replication in these cells.     

Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction

Functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4(+) and CD8(+) T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4(+) T cells. To investigate the role of PD-1 in HIV-associated CD4(+) T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-gamma-producing HIV-specific CD4(+) T cells compared with total or CMV-specific CD4(+) T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4(+) T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4(+) T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4(+) T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4(+) T cells.