Matrix Protein and Another Viral Component Contribute to Induction of Apoptosis in Cells Infected with Vesicular Stomatitis Virus

The induction of apoptosis in host cells is a prominent cytopathic effect of vesicular stomatitis virus (VSV) infection. The viral matrix (M) protein is responsible for several important cytopathic effects, including the inhibition of host gene expression and the induction of cell rounding in VSV-infected cells. This raises the question of whether M protein is also involved in the induction of apoptosis. HeLa or BHK cells were transfected with M mRNA to determine whether M protein induces apoptosis when expressed in the absence of other viral components. Expression of M protein induced apoptotic morphological changes and activated caspase-3 in both cell types, indicating that M protein induces apoptosis in the absence of other viral components. An M protein containing a point mutation that renders it defective in the inhibition of host gene expression (M51R mutation) activated little, if any, caspase-3, while a deletion mutant lacking amino acids 4 to 21 that is defective in the virus assembly function but fully functional in the inhibition of host gene expression was as effective as wild-type (wt) M protein in activating caspase-3. To determine whether M protein influences the induction of apoptosis in the context of a virus infection, the M51R M protein mutation was incorporated onto a wt background by using a recombinant infectious cDNA clone (rM51R-M virus). The timing of the induction of apoptosis by rM51R-M virus was compared to that by the corresponding recombinant wt (rwt) virus and to that by tsO82 virus, the mutant virus in which the M51R mutation was originally identified. In HeLa cells, rwt virus induced apoptosis faster than did rM51R-M virus, demonstrating a role for M protein in the induction of apoptosis. In contrast to the results obtained with HeLa cells, rwt virus induced apoptosis more slowly than did rM51R-M virus in BHK cells. This indicates that a viral component other than M protein contributes to induction of apoptosis in BHK cells and that wt M protein acts to delay induction of apoptosis by the other viral component. tsO82 virus induced apoptosis more rapidly than did rM51R-M virus in both HeLa and BHK cells. These two viruses contain the same point mutation in their M proteins, suggesting that sequence differences in genes other than that for M protein affect their rates of induction of apoptosis.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Contrasting effects of matrix protein on apoptosis in HeLa and BHK cells infected with vesicular stomatitis virus are due to inhibition of host gene expression

Vesicular stomatitis virus (VSV) is a potent inducer of apoptosis in host cells. Recently, it has been shown that two VSV products are involved in the induction of apoptosis, the matrix (M) protein, and another viral product that has yet to be identified (S. A. Kopecky et. al., J. Virol. 75:12169-12181, 2001). Comparison of recombinant viruses containing wild-type (wt) or mutant M proteins showed that wt M protein accelerates VSV-induced apoptosis in HeLa cells, while wt M protein delays apoptosis in VSV-infected BHK cells. Our hypothesis to explain these results is that both effects of M protein are due to the ability of M protein to inhibit host gene expression. This hypothesis was tested by infecting cells with an M protein mutant virus defective in the inhibition of host gene expression (rM51R-M virus) in the presence or absence of actinomycin D, another inhibitor of host gene expression. Actinomycin D accelerated induction of apoptosis of HeLa cells infected with rM51R-M virus and delayed apoptosis in BHK cells infected with rM51R-M virus, similar to the effects of wt M protein. The idea that the induction of apoptosis by M protein in HeLa cells is due to its ability to inhibit host gene expression was further tested by comparing the activation of upstream caspase pathways by M protein versus that by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB). Expression of M protein activated both caspase-8 and caspase-9-like enzymes, as did treatment with actinomycin D or DRB. Induction of apoptosis by M protein, actinomycin D, and DRB was inhibited in stably transfected HeLa cell lines that overexpress Bcl-2, an antiapoptotic protein that inhibits the caspase-9 pathway. A synthetic inhibitor of caspase-8, Z-IETD-FMK, did not inhibit induction of apoptosis by M protein, actinomycin D, or DRB. Taken together, our data support the hypothesis that the induction of apoptosis by M protein is caused by the inhibition of host gene expression and that the caspase-9 pathway is more important than the caspase-8 pathway for the induction of apoptosis by M protein and other inhibitors of host gene expression.     

Gene expression and antiviral activity of alpha/beta interferons and interleukin-29 in virus-infected human myeloid dendritic cells

Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-alpha), alpha/beta interferon (IFN-alpha/beta), and IFN-like interleukin-28A/B (IFN-lambda2/3) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-alpha, IFN-alpha/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-alpha, IFN-alpha, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-alpha. IFN-alpha priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-alpha or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.     

Cytokine and chemokine expression profiles of maturing dendritic cells using multiprotein platform arrays

Understanding the whole process of dendritic cell (DC) activation might help in the development of more efficient immunotherapeutic strategies for tumor patients. Part of this process is cytokine secretion, which has important effects on innate and adaptive immune response. Here, we cultured circulating monocytes for five days with interleukin-4 and GM-CSF followed by two-day culture with or without CD40 ligand and LPS to create a mature DC (mDC) and an immature DC (iDC) phenotype, respectively, characterized by differential expression of co-stimulatory molecules (CD80, CD83). We then compared the cytokine expression profile of the mDC and iDC using two protein platform arrays. Twelve supernatants from mDC paired with 12 from iDC were compared. The mDC protein expression profile showed significant increases in 16 out of 34 factors tested, including TNFalpha, IL-10, IL-12, IFNgamma, MIP1alpha, MIP1beta, IL-8, MDC, RANTES, and IL-6, which play a crucial role in the regulation of the innate immune response as well as the recruitment and activation of adaptive immune effectors. Interestingly, some of the cytokines expressed during maturation were also found in the gene expression profile identified in tumor metastases following IL-2 therapy using cDNA arrays. This finding suggests a possible role for resident DC maturation as a mediator of systemic IL-2 effects. Most important, the array of cytokines secreted during DC maturation may be considered an important component during adoptive transfer. Further characterization of the kinetics and persistence of their secretion should be undertaken in the future.     

Regulation of antigen presentation machinery in human dendritic cells by recombinant adenovirus

Recombinant adenoviral vectors (AdV) are potent vehicles for antigen engineering of dendritic cells (DC). DC engineered with AdV to express full length tumor antigens are capable stimulators of antigen-specific polyclonal CD8+ and CD4+ T cells. To determine the impact of AdV on the HLA class I antigen presentation pathway, we investigated the effects of AdV transduction on antigen processing machinery (APM) components in human DC. Interactions among AdV transduction, maturation, APM regulation and T cell activation were investigated. The phenotype and cytokine profile of DC transduced with AdV was intermediate, between immature (iDC) and matured DC (mDC). Statistically significant increases in expression were observed for peptide transporters TAP-1 and TAP-2, and HLA class I peptide-loading chaperone ERp57, as well as co-stimulatory surface molecule CD86 due to AdV transduction. AdV transduction enhanced the expression of APM components and surface markers on mDC, and these changes were further modulated by the timing of DC maturation. Engineering of matured DC to express a tumor-associated antigen stimulated a broader repertoire of CD8+ T cells, capable of recognizing immunodominant and subdominant epitopes. These data identify molecular changes in AdV-transduced DC (AdV/DC) that could influence T cell priming and should be considered in design of cancer vaccines.     

Immune response in the absence of neurovirulence in mice infected with m protein mutant vesicular stomatitis virus

Matrix (M) protein mutants of vesicular stomatitis virus (VSV), such as rM51R-M virus, are less virulent than wild-type (wt) VSV strains due to their inability to suppress innate immunity. Studies presented here show that when inoculated intranasally into mice, rM51R-M virus was cleared from nasal mucosa by day 2 postinfection and was attenuated for spread to the central nervous system, in contrast to wt VSV, thus accounting for its reduced virulence. However, it stimulated an antibody response similar to that in mice infected with the wt virus, indicating that it has the ability to induce adaptive immunity in vivo without causing disease. These results support the use of M protein mutants of VSV as vaccine vectors.     

Hemozoin (malarial pigment) inhibits differentiation and maturation of human monocyte-derived dendritic cells: a peroxisome proliferator-activated receptor-gamma-mediated effect

Acute and chronic Plasmodium falciparum malaria are accompanied by severe immunodepression possibly related to subversion of dendritic cells (DC) functionality. Phagocytosed hemozoin (malarial pigment) was shown to inhibit monocyte functions related to immunity. Hemozoin-loaded monocytes, frequently found in circulation and adherent to endothelia in malaria, may interfere with DC development and play a role in immunodepression. Hemozoin-loaded and unloaded human monocytes were differentiated in vitro to immature DC (iDC) by treatment with GM-CSF and IL-4, and to mature DC (mDC) by LPS challenge. In a second setting, hemozoin was fed to iDC further cultured to give mDC. In both settings, cells ingested large amounts of hemozoin undegraded during DC maturation. Hemozoin-fed monocytes did not apoptose but their differentiation and maturation to DC was severely impaired as shown by blunted expression of MHC class II and costimulatory molecules CD83, CD80, CD54, CD40, CD1a, and lower levels of CD83-specific mRNA in hemozoin-loaded iDC and mDC compared with unfed or latex-loaded DC. Further studies indicated activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in hemozoin-loaded iDC and mDC, associated with increased expression of PPAR-gamma mRNA, without apparent involvement of NF-kappaB. Moreover, expression of PPAR-gamma was induced and up-regulation of CD83 was inhibited by supplementing iDC and mDC with plausible concentrations of 15(S)-hydroxyeicosatetraenoic acid, a PPAR-gamma ligand abundantly produced by hemozoin via heme-catalyzed lipoperoxidation.     

Monocyte-derived CD1a+ and CD1a- dendritic cell subsets differ in their cytokine production profiles, susceptibilities to transfection, and capacities to direct Th cell differentiation

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-gamma, but they remained CD1a- and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.     

Primary human mDC1, mDC2, and pDC dendritic cells are differentially infected and activated by respiratory syncytial virus

Respiratory syncytial virus (RSV) causes recurrent infections throughout life. Vaccine development may depend upon understanding the molecular basis for induction of ineffective immunity. Because dendritic cells (DCs) are critically involved in early responses to infection, their interaction with RSV may determine the immunological outcome of RSV infection. Therefore, we investigated the ability of RSV to infect and activate primary mDCs and pDCs using recombinant RSV expressing green fluorescent protein (GFP). At a multiplicity of infection of 5, initial studies demonstrated ～6.8% of mDC1 and ～0.9% pDCs were infected. We extended these studies to include CD1c(-)CD141(+) mDC2, finding mDC2 infected at similar frequencies as mDC1. Both infected and uninfected cells upregulated phenotypic markers of maturation. Divalent cations were required for infection and maturation, but maturation did not require viral replication. There is evidence that attachment and entry/replication processes exert distinct effects on DC activation. Cell-specific patterns of RSV-induced maturation and cytokine production were detected in mDC1, mDC2, and pDC. We also demonstrate for the first time that RSV induces significant TIMP-2 production in all DC subsets. Defining the influence of RSV on the function of selected DC subsets may improve the likelihood of achieving protective vaccine-induced immunity.     

Increased pressure stimulates aberrant dendritic cell maturation

Patients with malignancy typically exhibit abnormal dendritic cell profiles. Interstitial tumor pressure is increased 20-50mmHg over that in normal tissue. We hypothesized that elevated pressure in the tumor microenvironment may influence dendritic cell (DC) phenotype and function. Monocyte-derived immature and mature DC isolated from healthy human donors were exposed to either ambient or 40 mmHg increased pressure at 37°C for 12 hours, then assessed for expression of CD80, CD86, CD83, CD40, MHC-I and MHC-II. IL-12 production and phagocytosis of CFSE-labeled tumor lysate were assessed in parallel. Elevated pressure significantly increased expression of all co-stimulatory and MHC molecules on mature DC. Immature DC significantly increased expression of CD80, CD86, CD83 and MHC-II, but not MHC-I and CD40, versus ambient pressure controls. Pressure-treated immature DC phenotypically resembled mature DC controls, but produced low IL-12. Phenotypic maturation correlated with decreased phagocytic capacity. These results suggest increased extracellular pressure may cause aberrant DC maturation and impair tumor immunosurveillance.     

Rapid Differentiation of Monocytes into Type I IFN-Producing Myeloid Dendritic Cells as an Antiviral Strategy against Influenza Virus Infection

Myeloid dendritic cells (mDCs) have long been thought to function as classical APCs for T cell responses. However, we demonstrate that influenza viruses induce rapid differentiation of human monocytes into mDCs. Unlike the classic mDCs, the virus-induced mDCs failed to upregulate DC maturation markers and were unable to induce allogeneic lymphoproliferation. Virus-induced mDCs secreted little, if any, proinflammatory cytokines; however, they secreted a substantial amount of chemoattractants for monocytes (MCP-1 and IP-10). Interestingly, the differentiated mDCs secreted type I IFN and upregulated the expression of IFN-stimulated genes (tetherin, IFITM3, and viperin), as well as cytosolic viral RNA sensors (RIG-I and MDA5). Additionally, culture supernatants from virus-induced mDCs suppressed the replication of virus in vitro. Furthermore, depletion of monocytes in a mouse model of influenza infection caused significant reduction of lung mDC numbers, as well as type I IFN production in the lung. Consequently, increased lung virus titer and higher mortality were observed. Taken together, our results demonstrate that the host responds to influenza virus infection by initiating rapid differentiation of circulating monocytes into IFN-producing mDCs, which contribute to innate antiviral immune responses.     

Antiviral-activated dendritic cells: a paracrine-induced response state

Infection of immature dendritic cells (DCs) by virus stimulates their maturation into APC. Infected DCs can also expose uninfected DCs to a panoply of cytokines/chemokines via paracrine signaling. Mathematical modeling suggests that a high rate of paracrine signaling is likely to occur among DCs located in three-dimensional space. Relatively little is known about how secreted factors modify the early response to virus infection. We used a transwell experimental system that allows passage of secreted factors, but not direct contact, between virus-infected DCs and uninfected DCs to investigate paracrine signaling responses. Paracrine signaling from infected DCs induced an antiviral-primed DC state distinct from that of mature virus-infected DCs that we refer to as antiviral-activated DCs (AVDCs). AVDCs had increased surface MHC class II and CD86 levels, but in contrast to virus-infected DCs, their MHC class I levels were unchanged. Imaging flow cytometry showed that AVDCs had an increased rate of phagocytosis compared with naive DCs. Experiments with IFN-beta cytokine indicated that it may be responsible for CD86, but not MHC class II regulation in AVDCs. Both IFN-inducible and IFN-independent genes are up-regulated in AVDCs. Notably, AVDCs are relatively resistant to virus infection in comparison to naive DCs and achieve accelerated and augmented levels of costimulatory molecule expression with virus infection. AVDCs show a distinct antiviral-primed state of DC maturation mediated by DC paracrine signaling. Although further in vivo study is needed, the characteristics of the AVDC suggest that it is well suited to play a role in the early innate-adaptive transition of the immune system.     

Dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins

Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational modification of proteins and has been implicated in multiple aspects of the immune response. To investigate the involvement of glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human monocyte-derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-glycans. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant lectins and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins galectin-3 and -8 increased during maturation and up-regulation of sialic acid expression by mDC correlated with an increased binding of siglec-1, -2, and -7.     

Human BDCA-1-positive blood dendritic cells differentiate into phenotypically distinct immature and mature populations in the absence of exogenous maturational stimuli: differentiation failure in HIV infection

Current immunological opinion holds that myeloid dendritic cell (mDC) precursors migrate from the blood to the tissues, where they differentiate into immature dermal- and Langerhans-type dendritic cells (DC). Tissue DC require appropriate signals from pathogens or inflammatory cytokines to mature and migrate to secondary lymphoid tissue. We show that purified blood mDC cultured in vitro with GM-CSF and IL-4, but in the absence of added exogenous maturation stimuli, rapidly differentiate into two maturational and phenotypically distinct populations. The major population resembles immature dermal DC, being positive for CD11b, CD1a, and DC-specific ICAM-3-grabbing nonintegrin. They express moderate levels of MHC class II and low levels of costimulatory molecules. The second population is CD11b(-/low) and lacks CD1a and DC-specific ICAM-3-grabbing nonintegrin but expresses high levels of MHC class II and costimulatory molecules. Expression of CCR7 on the CD11b(-/low) population and absence on the CD11b(+) cells further supports the view that these cells are mature and immature, respectively. Differentiation into mature and immature populations was not blocked by polymyxin B, an inhibitor of LPS. Neither population labeled for Langerin, E-cadherin, or CCR6 molecules expressed by Langerhans cells. Stimulation of 48-h cultured DC with LPS, CD40L, or poly(I:C) caused little increase in MHC or costimulatory molecule expression in the CD11b(-/low) DC but caused up-regulated expression in the CD11b(+) cells. In HIV-infected individuals, there was a marked decrease in the viability of cultured blood mDC, a failure to differentiate into the two populations described for normal donors, and an impaired ability to stimulate T cell proliferation.     

Influenza virus evades innate and adaptive immunity via the NS1 protein

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.     

Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-beta was higher in mDC than the profile of IFN-alpha. Wild-type IC induced markedly high levels of IFN-beta compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)-/- mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.     

The reciprocal interaction of NK cells with plasmacytoid or myeloid dendritic cells profoundly affects innate resistance functions

A reciprocal activating interaction between NK cells and dendritic cells (DC) has been suggested to play a role in the functional regulation of these cells in immunity, but it has been studied only using in vitro generated bone marrow- or monocyte-derived DC. We report that human peripheral blood plasmacytoid DC (pDC) and myeloid DC are necessary to induce NK cell function depending on the type of microbial stimulus. pDC and myeloid DC are required for strongly increased NK cytolytic activity and CD69 expression, in response to inactivated influenza virus or CpG-containing oligonucleotides and poly(I:C), respectively. Secreted type I IFN is required and sufficient for the augmentation of NK cell cytolytic activity in the coculture with pDC or myeloid DC, whereas CD69 expression is dependent on both type I IFN and TNF. In addition, in response to poly(I:C), myeloid DC induce NK cells to produce IFN-gamma through a mechanism dependent on both IL-12 secretion and cell contact between NK cells and myeloid DC, but independent of type I IFN. IL-2-activated NK cells have little to no cytolytic activity for immature myeloid DC and pDC, but are able to induce maturation of these cells. Moreover, IL-2-activated NK cells induce, in the presence of a suboptimal concentration of CpG-containing oligonucleotides, a strong IFN-alpha and TNF production. These data suggest that the reciprocal functional interaction between NK cells and either pDC or myeloid DC may play an important physiological role in the regulation of both innate resistance and adaptive immunity to infections.