Alternate Reading Frame Protein (F Protein) of Hepatitis C Virus: Paradoxical Effects of Activation and Apoptosis on Human Dendritic Cells Lead to Stimulation of T Cells

Hepatitis C virus (HCV) leads to chronic infection in the majority of infected individuals due to lack, failure, or inefficiency of generated adaptive immune responses. In a minority of patients, acute infection is followed by viral clearance. The immune correlates of viral clearance are not clear yet but have been extensively investigated, suggesting that multispecific and multifunctional cellular immunity is involved. The generation of cellular immunity is highly dependent upon how antigen presenting cells (APCs) process and present various viral antigens. Various structural and non-structural HCV proteins derived from the open reading frame (ORF) have been implicated in modulation of dendritic cells (DCs) and APCs. Besides the major ORF proteins, the HCV core region also encodes an alternate reading frame protein (ARFP or F), whose function in viral pathogenesis is not clear. In the current studies, we sought to determine the role of HCV-derived ARFP in modulating dendritic cells and stimulation of T cell responses. Recombinant adenovirus vectors containing F or core protein derived from HCV (genotype 1a) were prepared and used to endogenously express these proteins in dendritic cells. We made an intriguing observation that endogenous expression of F protein in human DCs leads to contrasting effects on activation and apoptosis of DCs, allowing activated DCs to efficiently internalize apoptotic DCs. These in turn result in efficient ability of DCs to process and present antigen and to prime and stimulate F protein derived peptide-specific T cells from HCV-naive individuals. Taken together, our findings suggest important aspects of F protein in modulating DC function and stimulating T cell responses in humans.     

Baseline viral load and immune activation determine the extent of reconstitution of innate immune effectors in HIV-1-infected subjects undergoing antiretroviral treatment

We analyzed dendritic cell (DC) and NK cell compartments in relation to CD4 recovery in 21 HIV-infected subjects followed to <50 copies/ml once starting antiretroviral therapy (ART) and observed for 52 wk of sustained suppression. Although CD4 counts increased in all subjects in response to ART, we observed a restoration of functional plasmacytoid DC (PDC) after 52 wk of sustained suppression under ART (from 1850 cells/ml to 4550 cells/ml) to levels comparable to controls (5120 cells/ml) only in subjects with a low baseline viral load, which also rapidly suppressed to <50 copies/ml upon 相似文献   

Dendritic cell–tumor cell hybrids and immunotherapy: what's next?

Dendritic cells (DC) are professional antigen-presenting cells currently being used as a cellular adjuvant in cancer immunotherapy strategies. Unfortunately, DC-based vaccines have not demonstrated spectacular clinical results. DC loading with tumor antigens and DC differentiation and activation still require optimization. An alternative technique for providing antigens to DC consists of the direct fusion of dendritic cells with tumor cells. These resulting hybrid cells may express both major histocompatibility complex (MHC) class I and II molecules associated with tumor antigens and the appropriate co-stimulatory molecules required for T-cell activation. Initially tested in animal models, this approach has now been evaluated in clinical trials, although with limited success. We summarize and discuss the results from the animal studies and first clinical trials. We also present a new approach to inducing hybrid formation by expression of viral fusogenic membrane glycoproteins.     

Targeting Viral Antigens to CD11c on Dendritic Cells Induces Retrovirus-Specific T Cell Responses

Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.     

Influenza A Virus Infection of Human Primary Dendritic Cells Impairs Their Ability to Cross-Present Antigen to CD8 T Cells

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection.     

Immunomodulatory Cross-Talk between Conjunctival Goblet Cells and Dendritic Cells

Goblet cells are secretory epithelial cells of mucosal tissues that confer protection from environmental agents or pathogens via expression and secretion of soluble mucins. Loss of these cells is associated with several chronic inflammatory disorders of the mucosa. Although demonstrated to transfer antigens from the luminal surface to stromal cells in the intestinal mucosa, it is not known if goblet cells contribute to the regulation of an immune response. In this study we report that similar to intestinal and respiratory mucosal epithelia, mouse ocular surface epithelia predominantly express the TGF-ß2 isoform. Specifically, we demonstrate the ability of goblet cells to express TGF-ß2 and increase it in response to Toll-Like Receptor 4 mediated stimulus in cultures. Goblet cells not only express TGF-ß2, but are also able to activate it in a thrombospondin-1 (TSP-1) dependent manner via their cell surface receptor CD36. Furthermore, goblet cell derived soluble factors that possibly include TGF-ß2, alter dendritic cell (DC) phenotype to a tolerogenic type by downregulating DC expression of MHC class II and co-stimulatory molecules CD80, CD86 and CD40. Thus our study demonstrates goblet cells as a cellular source of active TGF-ß2 in ocular mucosa and implicates their immunomodulatory function in maintaining mucosal immune homeostasis.     

Effects of lipofectin-antigen complexes on major histocompatibility complex class I-restricted antigen presentation pathway in murine dendritic cells and on dendritic cell maturation

We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF-antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.     

Persistent decreases in blood plasmacytoid dendritic cell number and function despite effective highly active antiretroviral therapy and increased blood myeloid dendritic cells in HIV-infected individuals

Dendritic cells (DC) have an instrumental role in the activation and function of both innate and adaptive immune responses. In humans, at least two distinct DC subsets have been characterized based on phenotypic markers: the myeloid DC (MDC) and the plasmacytoid DC (PDC). Both subsets are critical producers of cytokines (IL-12 for MDC and type I/II IFNs for PDC) and are functionally different. We show in this study that HIV(+) individuals have a significant decrease in the number of the Lin(-)HLA-DR(+)CD123(+) and BDCA-2(+) PDC compared with uninfected donors (p = 0.0001). HIV(+) individuals also have a sustained impairment in viral-induced IFN-alpha production (p < 0.0001). The decrease of the PDC subsets did not correlate with CD4 count or viral load and was not reversed in subjects under virally suppressive treatment, suggesting an irreversible change after infection. By contrast, the absolute number and median frequency of MDC in HIV-infected individuals were similar to those observed in uninfected controls, while a significant decrease was present in subjects with >5000 HIV-1 copies/ml. The inverse association with viral load of the MDC number, but not of IFN-alpha secretion or the number of PDC, suggests a role for MDC in viral control. Our data suggest that DC subsets are differentially reconstituted during the immune recovery associated with antiviral therapy. The persistent impairment of certain DC subsets may result in a sustained defect in DC-mediated innate immune functions despite an effective treatment regimen.     

A blunted blood plasmacytoid dendritic cell response to an acute systemic viral infection is associated with increased disease severity

At least two distinct human dendritic cell (DC) subsets are produced in the bone marrow and circulate in the peripheral blood-precursor myeloid DCs (pre-mDCs) and plasmacytoid DCs (PDCs). Both lineages of DCs are instrumental in antiviral innate immunity and shaping Th1 adaptive immune responses. PDCs are the most potent IFN-alpha-producing cells to viral pathogens. Dengue, an acute flavivirus disease, provides a model to study DC responses to a self-limited human viral infection. We analyzed circulating DC subsets in a prospective study of children with dengue across a broad range of illness severities: healthy controls; mild, nondengue, presumed viral infections; moderately ill dengue fever; and, the most severe form of illness, dengue hemorrhagic fever. We also examined PDC responses in monkeys with asymptomatic dengue viremia and to dengue virus exposure in vitro. The absolute number and frequency of circulating pre-mDCs early in acute viral illness decreased as illness severity increased. Depressed pre-mDC blood levels appeared to be part of the typical innate immune response to acute viral infection. The frequency of circulating PDCs trended upward and the absolute number of circulating PDCs remained stable early in moderately ill children with dengue fever, mild other, nondengue, febrile illness, and monkeys with asymptomatic dengue viremia. However, there was an early decrease in circulating PDC levels in children who subsequently developed dengue hemorrhagic fever. A blunted blood PDC response to dengue virus infection was associated with higher viremia levels, and was part of an altered innate immune response and pathogenetic cascade leading to severe disease.     

Receptor cross-linking on human plasmacytoid dendritic cells leads to the regulation of IFN-alpha production

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-alpha in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-alpha production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-alpha production at the level of IRF-7, while the decrease in IFN-alpha production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.     

Blockade of PD-1/PD-L1 Promotes Adoptive T-Cell Immunotherapy in a Tolerogenic Environment

Adoptive cellular immunotherapy using in vitro expanded CD8⁺ T cells shows promise for tumour immunotherapy but is limited by eventual loss of function of the transferred T cells through factors that likely include inactivation by tolerogenic dendritic cells (DC). The co-inhibitory receptor programmed death-1 (PD-1), in addition to controlling T-cell responsiveness at effector sites in malignancies and chronic viral diseases is an important modulator of dendritic cell-induced tolerance in naive T cell populations. The most potent therapeutic capacity amongst CD8⁺ T cells appears to lie within Tcm or Tcm-like cells but memory T cells express elevated levels of PD-1. Based on established trafficking patterns for Tcm it is likely Tcm-like cells interact with lymphoid-tissue DC that present tumour-derived antigens and may be inherently tolerogenic to develop therapeutic effector function. As little is understood of the effect of PD-1/PD-L1 blockade on Tcm-like CD8⁺ T cells, particularly in relation to inactivation by DC, we explored the effects of PD-1/PD-L1 blockade in a mouse model where resting DC tolerise effector and memory CD8⁺ T cells. Blockade of PD-1/PD-L1 promoted effector differentiation of adoptively-transferred Tcm-phenotype cells interacting with tolerising DC. In tumour-bearing mice with tolerising DC, effector activity was increased in both lymphoid tissues and the tumour-site and anti-tumour activity was promoted. Our findings suggest PD-1/PD-L1 blockade may be a useful adjunct for adoptive immunotherapy by promoting effector differentiation in the host of transferred Tcm-like cells.     

A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.     

Physiological State of Human Embryonic Lung Cells Affects Their Response to Human Cytomegalovirus

Cultures of human embryonic lung (HEL) cells in different physiological states were studied for their susceptibility to infection with human cytomegalovirus (CMV) with respect to production of infectious virus, synthesis of viral antigens, and virus-induced stimulation of cellular DNA synthesis. In general, subconfluent, actively growing cells yielded higher amounts of infectious virus than did confluent contact-inhibited cells. The higher yield of infectious virus was correlated with a greater percentage of cells producing viral antigens within the first 48 h after infection. In confluent cultures, 25 to 50% of the cells produced viral antigens within the first 48 h postinfection. This proportion did not change over a 10-fold range of multiplicity of infection, indicating that many of the cells in confluent cultures did not support productive infection. However, virtually all the cells in subconfluent cultures were susceptible. Also, in contrast to herpes simplex virus and pseudorabies virus, infectious CMV is not produced by cells treated with 5-fluorouracil and thymidine. Virus-induced stimulation of cellular DNA synthesis in cells infected at high multiplicities of infection could be detected only in confluent cultures, in which cellular DNA synthesis had been previously suppressed, but could not be detected in similarly treated cultures of subconfluent cells. The lack of detectable stimulation of cellular DNA synthesis in the latter was related to the fact that practically all the cells in the culture synthesized viral antigens within the first 48 h after infection, productive infection and detectable synthesis of cellular DNA being mutually exclusive.     

Induction of Potent Human Immunodeficiency Virus Type 1-Specific T-Cell-Restricted Immunity by Genetically Modified Dendritic Cells

A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4(+) and CD8(+) HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.     

Usp18 Driven Enforced Viral Replication in Dendritic Cells Contributes to Break of Immunological Tolerance in Autoimmune Diabetes

Infection with viruses carrying cross-reactive antigens is associated with break of immunological tolerance and induction of autoimmune disease. Dendritic cells play an important role in this process. However, it remains unclear why autoimmune-tolerance is broken during virus infection, but usually not during exposure to non-replicating cross-reactive antigens. Here we show that antigen derived from replicating virus but not from non-replicating sources undergoes a multiplication process in dendritic cells in spleen and lymph nodes. This enforced viral replication was dependent on Usp18 and was essential for expansion of autoreactive CD8⁺ T cells. Preventing enforced virus replication by depletion of CD11c⁺ cells, genetically deleting Usp18, or pharmacologically inhibiting of viral replication blunted the expansion of autoreactive CD8⁺ T cells and prevented autoimmune diabetes. In conclusion, Usp18-driven enforced viral replication in dendritic cells can break immunological tolerance and critically influences induction of autoimmunity.     

Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.     

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

Virus-specific CD8⁺ T cells (T_CD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T_CD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T_CD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T_CD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines.     

Efficient uptake and rapid degradation of plasmid DNA by murine dendritic cells via a specific mechanism

In spite of the important roles of dendritic cells in DNA-based therapies, the cellular uptake mechanism of plasmid DNA (pDNA) in dendritic cells is poorly understood. The present study was undertaken to investigate the binding and uptake of pDNA in vitro using a murine dendritic cell line, DC2.4 cells. A significant and time-dependent cellular association of [32P]pDNA with DC2.4 cells was observed at 37 degrees C and this fell markedly at 4 degrees C. The binding and uptake of [32P]pDNA were significantly inhibited by cold pDNA, polyinosinic acid (poly[I]), dextran sulfate, or heparin, but not by polycytidylic acid (poly[C]), dextran, or EDTA, suggesting that a specific mechanism mediated by a receptor like the macrophage scavenger receptor may be involved. The TCA precipitation experiments showed that DC2.4 cells rapidly endocytosed and degraded a significant amount of [32P]pDNA at 37 degrees C and released the degradation products into the medium. The pDNA degradation was also significantly inhibited by poly[I], but not poly[C]. The rate of pDNA degradation by DC2.4 cells was significantly higher than that by macrophages. A confocal microscopic study using fluorescein-labeled pDNA confirmed the rapid internalization and degradation of pDNA by the dendritic cells. Taken together, these results indicate that pDNA is efficiently taken up and rapidly digested by the dendritic cells via a specific mechanism. These findings may suggest the important role of the dendritic cells in the innate immune system for host defense.