Efficient human cytomegalovirus reactivation is maturation dependent in the Langerhans dendritic cell lineage and can be studied using a CD14+ experimental latency model

Studies from a number of laboratories have shown that the myeloid lineage is prominent in human cytomegalovirus (HCMV) latency, reactivation, dissemination, and pathogenesis. Existing as a latent infection in CD34(+) progenitors and circulating CD14(+) monocytes, reactivation is observed upon differentiation to mature macrophage or dendritic cell (DC) phenotypes. Langerhans' cells (LCs) are a subset of periphery resident DCs that represent a DC population likely to encounter HCMV early during primary infection. Furthermore, we have previously shown that CD34(+) derived LCs are a site of HCMV reactivation ex vivo. Accordingly, we have utilized healthy-donor CD34(+) cells to study latency and reactivation of HCMV in LCs. However, the increasing difficulty acquiring healthy-donor CD34(+) cells--particularly from seropositive donors due to the screening regimens used--led us to investigate the use of CD14(+) monocytes to generate LCs. We show here that CD14(+) monocytes cultured with transforming growth factor β generate Langerin-positive DCs (MoLCs). Consistent with observations using CD34(+) derived LCs, only mature MoLCs were permissive for HCMV infection. The lytic infection of mature MoLCs is productive and results in a marked inhibition in the capacity of these cells to promote T cell proliferation. Pertinently, differentiation of experimentally latent monocytes to the MoLC phenotype promotes reactivation in a maturation and interleukin-6 (IL-6)-dependent manner. Intriguingly, however, IL-6-mediated effects were restricted to mature LCs, in contrast to observations with classical CD14(+) derived DCs. Consequently, elucidation of the molecular basis behind the differential response of the two DC subsets should further our understanding of the fundamental mechanisms important for reactivation.     

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.     

Gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons

We recently demonstrated that CD8(+) T cells could block herpes simplex virus type 1 (HSV-1) reactivation from latency in ex vivo trigeminal ganglion (TG) cultures without destroying the infected neurons. Here we establish that CD8(+) T-cell prevention of HSV-1 reactivation from latency is mediated at least in part by gamma interferon (IFN-gamma). We demonstrate that IFN-gamma was produced in ex vivo cultures of dissociated latently infected TG by CD8(+) T cells that were present in the TG at the time of excision. Depletion of CD8(+) T cells or neutralization of IFN-gamma significantly enhanced the rate of HSV-1 reactivation from latency in TG cultures. When TG cultures were treated with acyclovir for 4 days to insure uniform latency, supplementation with recombinant IFN-gamma blocked HSV-1 reactivation in 80% of cultures when endogenous CD8(+) T cells were present and significantly reduced and delayed HSV-1 reactivation when CD8(+) T cells or CD45(+) cells were depleted from the TG cultures. The effectiveness of recombinant IFN-gamma in blocking HSV-1 reactivation was lost when its addition to TG cultures was delayed by more than 24 h after acyclovir removal. We propose that when the intrinsic ability of neurons to inhibit HSV-1 gene expression is compromised, HSV-specific CD8(+) T cells are rapidly mobilized to produce IFN-gamma and perhaps other antiviral cytokines that block the viral replication cycle and maintain the viral genome in a latent state.     

Differential regulation of IFN-gamma, TNF-alpha, and IL-10 production by CD4(+) alphabetaTCR+ T cells and vdelta2(+) gammadelta T cells in response to monocytes infected with Mycobacterium tuberculosis-H37Ra.

Mycobacterium tuberculosis bacilli readily activate CD4(+) and gammadelta T cells. CD4(+) and gammadelta T cells were compared for their ability to regulate IFN-gamma, TNF-alpha, and IL-10 production, cytokines with significant roles in the immune response to M. tuberculosis. PBMC from healthy tuberculin positive donors were stimulated with live M. tuberculosis-H37Ra. CD4(+) and gammadelta T cells were purified by negative selection and tested in response to autologous monocytes infected with M. tuberculosis. Both subsets produced equal amounts of secreted IFN-gamma. However, the precursor frequency of IFN-gamma secreting gammadelta T cells was half that of CD4(+) T cells, indicating that gammadelta T cells were more efficient producers of IFN-gamma than CD4(+) T cells. TNF-alpha production was markedly enhanced by addition of CD4(+) and gammadelta T cells to M. tuberculosis infected monocytes, and TNF-alpha was produced by both T cells and monocytes. No differences in TNF-alpha enhancement were noted between CD4(+) and gammadelta T cells. IL-10 production by M. tuberculosis infected monocytes was not modulated by CD4(+) or gammadelta T cells. Thus CD4(+) and gammadelta T cells had similar roles in differential regulation of IFN-gamma, TNF-alpha, and IL-10 secretion in response to M. tuberculosis infected monocytes. However, the interaction between T cells and infected monocytes differed for each cytokine. IFN-gamma production was dependent on antigen presentation and costimulators provided by monocytes. TNF-alpha levels were increased by addition of TNF-alpha produced by T cells and IL-10 production by monocytes was not modulated by CD4(+) or gammadelta T cells.     

Evidence for Human Immunodeficiency Virus Type 1 Replication In Vivo in CD14+ Monocytes and Its Potential Role as a Source of Virus in Patients on Highly Active Antiretroviral Therapy

In vitro studies show that human immunodeficiency virus type 1 (HIV-1) does not replicate in freshly isolated monocytes unless monocytes differentiate to monocyte-derived macrophages. Similarly, HIV-1 may replicate in macrophages in vivo, whereas it is unclear whether blood monocytes are permissive to productive infection with HIV-1. We investigated HIV-1 replication in CD14(+) monocytes and resting and activated CD4(+) T cells by measuring the levels of cell-associated viral DNA and mRNA and the genetic evolution of HIV-1 in seven acutely infected patients whose plasma viremia had been <100 copies/ml for 803 to 1,544 days during highly active antiretroviral therapy (HAART). HIV-1 DNA was detected in CD14(+) monocytes as well as in activated and resting CD4(+) T cells throughout the course of study. While significant variation in the decay slopes of HIV-1 DNA was seen among individual patients, viral decay in CD14(+) monocytes was on average slower than that in activated and resting CD4(+) T cells. Measurements of HIV-1 sequence evolution and the concentrations of unspliced and multiply spliced mRNA provided evidence of ongoing HIV-1 replication, more pronounced in CD14(+) monocytes than in resting CD4(+) T cells. Phylogenetic analyses of HIV-1 sequences indicated that after prolonged HAART, viral populations related or identical to those found only in CD14(+) monocytes were seen in plasma from three of the seven patients. In the other four patients, HIV-1 sequences in plasma and the three cell populations were identical. CD14(+) monocytes appear to be one of the potential in vivo sources of HIV-1 in patients receiving HAART.     

Gamma interferon blocks gammaherpesvirus reactivation from latency in a cell type-specific manner

Gammaherpesviruses are important pathogens whose lifelong survival in the host depends critically on their capacity to establish and reactivate from latency, processes regulated by both viral genes and the host immune response. Previous work has demonstrated that gamma interferon (IFN-gamma) is a key regulator of chronic infection with murine gammaherpesvirus 68 (gammaHV68), a virus that establishes latent infection in B lymphocytes, macrophages, and dendritic cells. In mice deficient in IFN-gamma or the IFN-gamma receptor, gammaHV68 gene expression is altered during chronic infection, and peritoneal cells explanted from these mice reactivate more efficiently ex vivo than cells derived from wild-type mice. Furthermore, treatment with IFN-gamma inhibits reactivation of gammaHV68 from latently infected wild-type peritoneal cells, and depletion of IFN-gamma from wild-type mice increases the efficiency of reactivation of explanted peritoneal cells. These profound effects of IFN-gamma on chronic gammaHV68 latency and reactivation raise the question of which cells respond to IFN-gamma to control chronic gammaHV68 infection. Here, we show that IFN-gamma inhibited reactivation of peritoneal cells and spleen cells harvested from mice lacking B lymphocytes, but not wild-type spleen cells, suggesting that IFN-gamma may inhibit reactivation in a cell type-specific manner. To directly test this hypothesis, we expressed the diphtheria toxin receptor specifically on either B lymphocytes or macrophages and used diphtheria toxin treatment to deplete these specific cells in vivo and in vitro after establishing latency. We demonstrate that macrophages, but not B cells, are responsive to IFN-gamma-mediated suppression of gammaHV68 reactivation. These data indicate that the regulation of gammaherpesvirus latency by IFN-gamma is cell type specific and raise the possibility that cell type-specific immune deficiency may alter latency in distinct and important ways.     

Circulating Dendritic Cells Isolated from Healthy Seropositive Donors Are Sites of Human Cytomegalovirus Reactivation In Vivo

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34⁺ progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.     

Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-alpha and DM-alpha. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.     

Rates of reactivation of latent herpes simplex virus from mouse trigeminal ganglia ex vivo correlate directly with viral load and inversely with number of infiltrating CD8+ T cells

Herpes simplex viruses (HSV) reactivate at rates proportional to the viral loads in latently infected ganglia. However, these rates vary substantially among infected animals. We assessed whether the numbers of HSV-specific CD8(+) T cells infiltrating latently infected ganglia also affect reactivation rates and contribute to their variability. Following corneal infection of mice with HSV type 2 (HSV-2), we quantified the latent viral loads in dissociated trigeminal ganglia by real-time PCR, the numbers of infiltrating CD8(+) T cells by flow cytometry, and the rates of reactivation by the detection of cell-free virus released from ganglion cells cultured in 96-well plates. The reactivation rates correlated directly with the latent viral loads (P = 0.001) but did so more strongly (P = 10(-7)) when cultures were depleted of CD8(+) T cells. Reactivation rates were reduced in a dose-dependent fashion by adding back ganglion CD8(+) T cells to the cultures (P = 0.003). We related the latent viral loads, numbers of CD8(+) T cells, and reactivation rates by mathematical equations. The rates of reactivation predicted from latent viral loads and numbers of infiltrating CD8(+) T cells in dissociated ganglia correlated with the observed rates of reactivation (P = 0.04). The reactivation of HSV-2 from ganglia ex vivo is determined both by the latent viral load and the number of infiltrating CD8(+) T cells.     

Human cytomegalovirus latent infection of myeloid cells directs monocyte migration by up-regulating monocyte chemotactic protein-1

Following primary infection, human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic cells from which it reactivates to cause serious disease in immunosuppressed patients such as allograft recipients. HCMV is a common cause of disease in newborns and transplant patients and has also been linked with vascular diseases such as primary and post-transplant arteriosclerosis. A major factor in the pathogenesis of vascular disease is the CC chemokine MCP-1. In this study, we demonstrate that granulocyte macrophage progenitors (GMPs) latently infected with HCMV significantly increased expression of MCP-1 and that this phenotype was dependent on infection with viable virus. Inhibitors of a subset of G(alpha) proteins and PI3K inhibited the up-regulation of MCP-1 in latently infected cultures, suggesting that the mechanism underlying this phenotype involves signaling through a G-protein coupled receptor. In GMPs infected with the low passage viral strain Toledo, up-regulated MCP-1 was restricted to a subset of myeloid progenitor cells expressing CD33, HLA-DR, and CD14 but not CD1a, CD15, or CD16, and the increase in MCP-1 was sufficient to enhance migration of CD14(+) monocytes to latently infected cells. Latent HCMV-mediated up-regulation of MCP-1 provides a mechanism by which HCMV may contribute to vascular disease during the latent phase of infection or facilitate dissemination of virus upon reactivation from latency.     

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.     

Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells

Dendritic cell (DC) migration from the site of infection to the site of T-cell priming is a crucial event in the generation of antiviral T-cell responses. Here we present to our knowledge the first functional evidence that human cytomegalovirus (HCMV) blocks the migration of infected monocyte-derived DCs toward lymphoid chemokines CCL19 and CCL21. DC migration is blocked by viral impairment of the chemokine receptor switch at the level of the expression of CCR7 molecules. The inhibition occurs with immediate-early-early kinetics, and viral interference with NF-kappaB signaling is likely to be at least partially responsible for the lack of CCR7 expression. DCs which migrate from the infected cultures are HCMV antigen negative, and consequently they do not stimulate HCMV-specific CD8(+) T cells, while CD4(+)-T-cell activation is not impaired. Although CD8(+) T cells can also be activated by alternative antigen presentation mechanisms, the spatial segregation of naive T cells and infected DCs seems a potent mechanism of delaying the generation of primary CD8(+)-T-cell responses and aiding early viral spread.     

Reduced expression of HLA class II molecules and Iinterleukin-10- and transforming growth factor beta1-independent suppression of T-cell proliferation in human cytomegalovirus-infected macrophage cultures

After a primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in myeloid lineage cells, and the virus has developed several mechanisms to avoid immune recognition and destruction of infected cells. In this study, we show that HCMV utilizes two different strategies to reduce the constitutive expression of HLA-DR, -DP, and -DQ on infected macrophages and that infected macrophages are unable to stimulate a specific CD4+ T-cell response. Downregulation of the HLA class II molecules was observed in 90% of the donor samples and occurred in two phases: at an early (1 day postinfection [dpi]) time point postinfection and at a late (4 dpi) time point postinfection. The early inhibition of HLA class II expression and antigen presentation was not dependent on active virus replication, since UV-inactivated virus induced downregulation of HLA-DR and inhibition of T-cell proliferation at 1 dpi. In contrast, the late effect required virus replication and was dependent on the expression of the HCMV unique short (US) genes US1 to -9 or US11 in 77% of the samples. HCMV-treated macrophages were completely devoid of T-cell stimulation capacity at 1 and 4 dpi. However, while downregulation of HLA class II expression was rather mild, a 66 to 90% reduction in proliferative T-cell response was observed. This discrepancy was due to undefined soluble factors produced in HCMV-infected cell cultures, which did not include interleukin-10 and transforming growth factor beta1. These results suggest that HCMV reduces expression of HLA class II molecules on HCMV-infected macrophages and inhibits T-cell proliferation by different distinct pathways.     

Ex vivo stimulation and expansion of both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells of human cytomegalovirus-seropositive blood donors by using a soluble recombinant chimeric protein, IE1-pp65

The transfer of anti-human cytomegalovirus (HCMV) effector T cells to allogeneic bone marrow recipients results in protection from HCMV disease associated with transplantation, suggesting the direct control of CMV replication by T cells. IE1 and pp65 proteins, both targets of CD4(+) and CD8(+) T cells, are considered the best candidates for immunotherapy and vaccine design against HCMV. In this report, we describe the purification of a 165-kDa chimeric protein, IE1-pp65, and its use for in vitro stimulation and expansion of anti-HCMV CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMC) of HCMV-seropositive donors. We demonstrate that an important proportion of anti-HCMV CD4(+) T cells was directed against IE1-pp65 in HCMV-seropositive donors and that the protein induced activation of HLA-DR3-restricted anti-IE1 CD4(+) T-cell clones, as assessed by gamma interferon (IFN-gamma) secretion and cytotoxicity. Moreover, soluble IE1-pp65 stimulated and expanded anti-pp65 CD8(+) T cells from PBMC of HLA-A2, HLA-B35, and HLA-B7 HCMV-seropositive blood donors, as demonstrated by cytotoxicity, intracellular IFN-gamma labeling, and quantitation of peptide-specific CD8(+) cells using an HLA-A2-peptide tetramer and staining of intracellular IFN-gamma. These results suggest that soluble IE1-pp65 may provide an alternative to infectious viruses used in current adoptive strategies of immunotherapy.     

Autocrine type I IFN and contact with endothelium promote the presentation of influenza A virus by monocyte-derived APC

Purified monocytes infected with influenza A virus do not become mature dendritic cells (DCs) and they present viral peptides poorly to autologous memory T cells. In this study, we investigated whether influenza A-infected monocytes matured to DCs with a high capacity to stimulate T cells when they were infected with influenza A virus in a model tissue setting wherein they were cocultured with endothelium grown on a type I collagen matrix. Intercellular interactions with endothelium strongly promoted the Ag-presenting capacity of monocyte-derived cells infected with influenza A virus, and the heterologous coculture system also enhanced production of IFN-alpha by monocytes in the absence of plasmacytoid cells. Production of IFN-alpha in the presence of endothelium correlated with monocyte differentiation to mature DCs and their ability to stimulate proliferation and IFN-gamma production by autologous T cells. Monocyte-derived cells that developed into migratory DCs promoted proliferation of influenza A virus-specific CD4(+) and CD8(+) cells, whereas those that developed into macrophages promoted proliferation of CD8(+) T cells only. This onset of APC activity could be partially blocked with Ab to the IFN-alphabeta receptor when monocytes were infected with UV-treated virus, but neutralizing this pathway was inconsequential when monocytes were infected with live virus. Thus, type I IFN and direct contact with endothelium promote development of influenza A virus-presenting activity in monocyte-derived cells in a setting in which this differentiation does not depend on plasmacytoid cells. However, when infected with live influenza virus, the role of type I IFN in mediating differentiation and Ag-presenting capacity is expendable, apparently due to other mechanisms of virus-mediated activation.     

Uncoupled anti-HIV and immune-enhancing effects when combining IFN-alpha and IL-7

Cytokine-based therapies have been examined for purging viral reservoirs and immunomodulation in HIV infection. However, single cytokines did not result in either HIV eradication or an efficient HIV-specific immune response. We hypothesize that cytokines with distinct biologic effects need to be combined for immunotherapy of HIV infection. In this study, we investigated the anti-HIV activity and immune-enhancing effects of the combination of IFN-alpha and IL-7. In human lymphocyte aggregate cultures infected ex vivo with the X4 HIV strain NL4-3, IFN-alpha/IL-7 potently inhibited HIV replication and preserved CD4(+) T cells, probably by up-regulating Bcl-2. IFN-alpha/IL-7 also strongly inhibited R5 HIV replication. Furthermore, in allogeneic MLRs, IFN-alpha/IL-7 increased T cell proliferation and IFN-gamma production. IFN-alpha alone also had strong anti-HIV activity, but neither preserved CD4(+) T cells nor increased T cell responses in MLRs. IL-7 alone maintained T cells and enhanced T cell activation in MLRs, but only moderately inhibited or increased HIV replication. Thus, coadministration of IFN-alpha/IL-7 combines the potent anti-HIV activity of IFN-alpha with the beneficial effects of IL-7 on T cell survival and function. We speculate that IFN-alpha will block viral replication, activate APCs, and up-regulate MHC molecules, thus allowing IL-7 to display its effects for generating an efficient immune response. In this scenario, the known reactivation of latent HIV by IL-7 may be advantageous.