Maturation of blood-derived dendritic cells enhances human immunodeficiency virus type 1 capture and transmission

Dendritic cells (DCs) are specialized antigen-presenting cells. However, DCs exposed to human immunodeficiency virus type 1 (HIV-1) are also able to transmit a vigorous cytopathic infection to CD4(+) T cells, a process that has been frequently related to the ability of DC-SIGN to bind HIV-1 envelope glycoproteins. The maturation of DCs can increase the efficiency of HIV-1 transmission through trans infection. We aimed to comparatively study the effect of maturation in monocyte-derived DCs (MDDCs) and blood-derived myeloid DCs during the HIV-1 capture process. In vitro capture and transmission of envelope-pseudotyped HIV-1 and its homologous replication-competent virus to susceptible target cells were assessed by p24(gag) detection, luciferase activity, and both confocal and electron microscopy. Maturation of MDDCs or myeloid DCs enhanced the active capture of HIV-1 in a DC-SIGN- and viral envelope glycoprotein-independent manner, increasing the life span of trapped virus. Moreover, higher viral transmission of mature DCs to CD4(+) T cells was highly dependent on active viral capture, a process mediated through cholesterol-enriched domains. Mature DCs concentrated captured virus in a single large vesicle staining for CD81 and CD63 tetraspanins, while immature DCs lacked these structures, suggesting different intracellular trafficking processes. These observations help to explain the greater ability of mature DCs to transfer HIV-1 to T lymphocytes, a process that can potentially contribute to the viral dissemination at lymph nodes in vivo, where viral replication takes place and there is a continuous interaction between susceptible T cells and mature DCs.     

Human immunodeficiency virus type 1 replication in dendritic cell-T-cell cocultures is increased upon incorporation of host LFA-1 due to higher levels of virus production in immature dendritic cells

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.     

Virus infection of dendritic cells: portal for host invasion and host defense

Dendritic cells (DCs) act as a portal for virus invasion and as the most potent antigen-presenting cells in antiviral host defense. Human immunodeficiency virus (HIV)-1 has served as the paradigm for virus interaction with DCs. HIV-1 infection of DCs via its primary CD4 receptor and secondary chemokine receptors leads to full virus replication (cis infection), whereas binding to C-type lectin receptors results both in cis replication, as well as transfer and replication of virus in CD4(pos) T cells (trans infection). DCs respond to this invasion by processing viral proteins through MHC class I and II pathways and undergoing a maturation that enhances their presentation of antigen to T cells for induction of adaptive antiviral immunity. HIV-1 and other viruses have evolved mechanisms to subvert this immune function. Engineering of DCs with various forms of viral immunogens and co-treatment with cytokines and chemokines is being used as an immunotherapy for HIV-1 and other viral infections.     

Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-alpha or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1alpha/beta and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.     

Endogenously expressed nef uncouples cytokine and chemokine production from membrane phenotypic maturation in dendritic cells

Immature dendritic cells (DCs), unlike mature DCs, require the viral determinant nef to drive immunodeficiency virus (SIV and HIV) replication in coculture with CD4(+) T cells. Since immature DCs may capture and get infected by virus during mucosal transmission, we hypothesized that Nef associated with the virus or produced during early replication might modulate DCs to augment virus dissemination. Adenovirus vectors expressing nef were used to introduce nef into DCs in the absence of other immunodeficiency virus determinants to examine Nef-induced changes that might activate immature DCs to acquire properties of mature DCs and drive virus replication. Nef expression by immature human and macaque DCs triggered IL-6, IL-12, TNF-alpha, CXCL8, CCL3, and CCL4 release, but without up-regulating costimulatory and other molecules characteristic of mature DCs. Coincident with this, nef-expressing immature DCs stimulated stronger autologous CD4(+) T cell responses. Both SIV and HIV nef-expressing DCs complemented defective SIVmac239 delta nef, driving replication in autologous immature DC-T cell cultures. In contrast, if DCs were activated after capturing delta nef, virus growth was not exacerbated. This highlights one way in which nef-defective virus-bearing immature DCs that mature while migrating to draining lymph nodes could induce stronger immune responses in the absence of overwhelming productive infection (unlike nef-containing wild-type virus). Therefore, Nef expressed in immature DCs signals a distinct activation program that promotes virus replication and T cell recruitment but without complete DC maturation, thereby lessening the likelihood that wild-type virus-infected immature DCs would activate virus-specific immunity, but facilitating virus dissemination.     

Opposite effects of IL-10 on the ability of dendritic cells and macrophages to replicate primary CXCR4-dependent HIV-1 strains

We investigated the effect of IL-10 on replication of primary CXCR4-dependent (X4) HIV-1 strains by monocyte-derived dendritic cells (DCs) and macrophages (M Phis). M Phis efficiently replicated CXCR4-dependent HIV-1 (X4 HIV-1) strains NDK and VN44, whereas low levels of p24 were detected in supernatants of infected DCs. IL-10 significantly increased X4 HIV-1 replication by DCs but blocked viral production by M Phis as determined by p24 levels and semiquantitative nested PCR. IL-10 up-regulated CXCR4 mRNA and protein expression on DCs and M Phis, suggesting that IL-10 enhances virus entry in DCs but blocks an entry and/or postentry step in M Phis. The effect of IL-10 on the ability of DCs and M Phis to transmit virus to autologous CD4(+) T lymphocytes was investigated in coculture experiments. DCs exhibited a greater ability than did M Phis to transmit a vigorous infection to CD4(+) T cells despite their very low replication capacity. IL-10 had no effect on HIV-1 replication in DC:T cell cocultures but markedly decreased viral production in M Phi:T cell cocultures. These results demonstrate that IL-10 has opposite effects on the replication of primary X4 HIV-1 strains by DCs and M Phis. IL-10 increases X4-HIV-1 replication in DCs but does not alter their capacity to transmit virus to CD4(+) T lymphocytes. These findings suggest that increased levels of IL-10 observed in HIV-1-infected patients with disease progression may favor the replication of X4 HIV-1 strains in vivo.     

Preferential infection of dendritic cells during human immunodeficiency virus type 1 infection of blood leukocytes

Human immunodeficiency virus type 1 (HIV-1) transmission by the parenteral route is similar to mucosal transmission in the predominance of virus using the CCR5 coreceptor (R5 virus), but it is unclear whether blood dendritic cells (DCs), monocytes, or T cells are the cells initially infected. We used ex vivo HIV-1 infection of sorted blood mononuclear cells to model the in vivo infection of blood leukocytes. Using quantitative real-time PCR to detect full-length HIV-1 DNA, both sorted CD11c⁺ myeloid and CD11c⁻ plasmacytoid DCs were more frequently infected than other blood mononuclear cells, including CD16⁺ or CD14⁺ monocytes or resting CD4⁺ T cells. There was a strong correlation between CCR5 coreceptor use and preferential DC infection across a range of HIV-1 isolates. After infection of unsorted blood mononuclear cells, HIV-1 was initially detected in the CD11c⁺ DCs and later in other leukocytes, including clustering DCs and activated T cells. DC infection with R5 virus was productive, as shown by efficient transmission to CD4⁺ T cells in coculture. Blood DCs infected with HIV-1 in vitro and cultured alone expressed only low levels of multiply spliced HIV-1 RNA unless cocultured with CD4⁺ T cells. Early selective infection of immature blood DCs by R5 virus and upregulation of viral expression during DC-T-cell interaction and transmission provide a potential pathway for R5 selection following parenteral transmission.     

Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

Background

Dendritic cells (DCs) are among the first cells to encounter HIV-1 and play important roles in viral transmission and pathogenesis. Immature DCs allow productive HIV-1 replication and long-term viral dissemination. The pro-inflammatory factor lipopolysaccharide (LPS) induces DC maturation and enhances the efficiency of DC-mediated HIV-1 transmission. Type I interferon (IFN) partially inhibits HIV-1 replication and cell-cell transmission in CD4⁺ T cells and macrophages. Tetherin is a type I IFN-inducible restriction factor that blocks HIV-1 release and modulates CD4⁺ T cell-mediated cell-to-cell transmission of HIV-1. However, the role of type I IFN and tetherin in HIV-1 infection of DCs and DC-mediated viral transmission remains unknown.

Results

We demonstrated that IFN-alpha (IFNα)-induced mature DCs restricted HIV-1 replication and trans-infection of CD4⁺ T cells. Tetherin expression in monocyte-derived immature DCs was undetectable or very low. High levels of tetherin were transiently expressed in LPS- and IFNα-induced mature DCs, while HIV-1 localized into distinct patches in these DCs. Knockdown of induced tetherin in LPS- or IFNα-matured DCs modestly enhanced HIV-1 transmission to CD4⁺ T cells, but had no significant effect on wild-type HIV-1 replication in mature DCs. Intriguingly, we found that HIV-1 replication in immature DCs induced significant tetherin expression in a Nef-dependent manner.

Conclusions

The restriction of HIV-1 replication and transmission in IFNα-induced mature DCs indicates a potent anti-HIV-1 response; however, high levels of tetherin induced in mature DCs cannot significantly restrict wild-type HIV-1 release and DC-mediated HIV-1 transmission. Nef-dependent tetherin induction in HIV-1-infected immature DCs suggests an innate immune response of DCs to HIV-1 infection.     

A fusion inhibitor prevents spread of immunodeficiency viruses, but not activation of virus-specific T cells, by dendritic cells

Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4(+) T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4(+), not CD8(+), T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4(+) T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4(+) and CD8(+) T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4(+) T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.     

Functionally distinct transmission of human immunodeficiency virus type 1 mediated by immature and mature dendritic cells

Dendritic cells (DCs) potently stimulate the transmission of human immunodeficiency virus type 1 (HIV-1) to CD4(+) T cells. Immature DCs (iDCs) located in submucosal tissues can capture HIV-1 and migrate to lymphoid tissues, where they become mature DCs (mDCs) for effective antigen presentation. DC maturation promotes HIV-1 transmission; however, the underlying mechanisms remain unclear. Here we have compared monocyte-derived iDCs and mDCs for their efficiencies and mechanisms of HIV-1 transmission. We have found that mDCs significantly facilitate HIV-1 endocytosis and efficiently concentrate HIV-1 at virological synapses, which contributes to mDC-enhanced viral transmission, at least in part. mDCs were more efficient than iDCs in transferring HIV-1 to various types of target cells independently of C-type lectins, which partially accounted for iDC-mediated HIV-1 transmission. Efficient HIV-1 trans-infection mediated by iDCs and mDCs required contact between DCs and target cells. Moreover, rapid HIV-1 degradation occurred in both iDCs and mDCs, which correlated with the lack of HIV-1 retention-mediated long-term viral transmission. Our results provide new insights into the mechanisms underlying DC-mediated HIV-1 transmission, suggesting that HIV-1 exploits mDCs to facilitate its dissemination within lymphoid tissues.     

Human seminal plasma abrogates the capture and transmission of human immunodeficiency virus type 1 to CD4+ T cells mediated by DC-SIGN

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is expressed by dendritic cells (DCs) at mucosal surfaces and appears to play an important role in the dissemination of human immunodeficiency virus type 1 (HIV-1) infection. DC-SIGN binds HIV-1 gp120 and efficiently transmits the virus to T CD4⁺ cells, which become the center of viral replication. Semen represents the main vector for HIV-1 dissemination worldwide. In the present study we show that human seminal plasma (SP), even when used at very high dilutions (1:10⁴ to 1:10⁵), markedly inhibits the capture and transmission of HIV-1 to T CD4⁺ cells mediated by both DCs and B-THP-1-DC-SIGN cells. In contrast, SP does not inhibit the capture of HIV-1 by DC-SIGN-negative target cells, such as the T-cell line SupT-1, monocytes, and activated peripheral blood mononuclear cells. The SP inhibitor has a high molecular mass (>100 kDa) and directly interacts with DC-SIGN-positive target cells but not with HIV-1. Moreover, the inhibitor binds to concanavalin A, suggesting that it contains high-mannose N-linked carbohydrates. Of note, using biotin-labeled SP we found that the binding of SP components to DCs was abrogated by mannan, while their interaction with B-THP-1 cells was almost completely dependent on the expression of DC-SIGN. Since epithelium integrity is often compromised after vaginal or anal intercourse, as well as in the presence of ulcerative-sexually transmitted diseases, our results support the notion that components of the SP might be able to access to the subepithelium, inhibiting the recognition of HIV-1 gp120 by DC-SIGN-positive DCs.     

HIV-1 selectively infects a subset of nonmaturing BDCA1-positive dendritic cells in human blood

The infection of cultured monocyte-derived dendritic cells (DCs) with HIV-1 involves CD4 and CCR5 receptors, while transmission to T cells is enhanced at least in part by the lectin DC-SIGN/CD209. In the present study, we studied BDCA-1+ myeloid DCs isolated directly from human blood. These cells express CD4 and low levels of CCR5 and CXCR4 coreceptors, but not DC-SIGN. The myeloid DCs replicate two R5 viruses, BaL and YU2, and transfer infection to activated T cells. The virus productively infects a small fraction of the blood DCs that fail to mature in culture, as indicated by the maturation markers CD83 and DC-LAMP/CD208, and the expression of high CD86 and MHC class II, in contrast to many noninfected DCs. A greater proportion of BDCA-1+ DCs are infected when the virus is pseudotyped with the vesicular stomatitis envelope VSV-G (5-15%), as compared with the R5 virus (0.3-3.5%), indicating that HIV-1 coreceptors may limit the susceptibility of DCs to become infected, or the endocytic route of viral entry used by HIV/vesicular stomatitis virus enhances infectivity. When infected and noninfected cells are purified by cell sorting, the former uniformly express HIV p24 gag and are virtually inactive as stimulators of the allogeneic MLR, in contrast to potent stimulation by noninfected DCs from the same cultures. These results point to two roles for a small fraction of blood DCs in HIV-1 pathogenesis: to support productive infection and to evade the direct induction of T cell-mediated immunity.     

HIV-1 N-glycan composition governs a balance between dendritic cell-mediated viral transmission and antigen presentation

The natural function of dendritic cells (DCs) is to capture and degrade pathogens for Ag presentation. However, HIV-1 can evade viral degradation by DCs and hijack DCs for migration to susceptible CD4(+) T lymphocytes. It is unknown what factors decide whether a virus is degraded or transmitted to T cells. The interaction of DCs with HIV-1 involves C-type lectin receptors, such as DC-specific ICAM-3-grabbing nonintegrin, which bind to the envelope glycoprotein complex (Env), which is decorated heavily with N-linked glycans. We hypothesized that the saccharide composition of the Env N-glycans is involved in avoiding viral degradation and Ag presentation, as well as preserving infectious virus for the transmission to target cells. Therefore, we studied the fate of normally glycosylated virus versus oligomannose-enriched virus in DCs. Changing the heterogeneous N-linked glycan composition of Env to uniform oligomannose N-glycans increased the affinity of HIV-1 for DC-specific ICAM-3-grabbing nonintegrin and enhanced the capture of HIV-1 by immature DCs; however, it decreased the subsequent transmission to target cells. Oligomannose-enriched HIV-1 was directed more efficiently into the endocytic pathway, resulting in enhanced viral degradation and reduced virus transfer to target cells. Furthermore, Env containing exclusively oligomannose N-glycans was presented to Env-specific CD4(+) T cells more efficiently. Taken together, our results showed that the HIV-1 N-glycan composition plays a crucial role in the balance between DC-mediated Ag degradation and presentation and DC-mediated virus transmission to target cells. This finding may have implications for the early events in HIV-1 transmission and the induction of antiviral immune responses.     

Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans-Infection Through Recognition of Viral Membrane Gangliosides

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4⁺ T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4⁺ T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.     

Lentivirus-mediated RNA interference of DC-SIGN expression inhibits human immunodeficiency virus transmission from dendritic cells to T cells

In the early events of human immunodeficiency virus type 1 (HIV-1) infection, immature dendritic cells (DCs) expressing the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) receptor capture small amounts of HIV-1 on mucosal surfaces and spread viral infection to CD4(+) T cells in lymph nodes (22, 34, 45). RNA interference has emerged as a powerful tool to gain insight into gene function. For this purpose, lentiviral vectors that express short hairpin RNA (shRNA) for the delivery of small interfering RNA (siRNA) into mammalian cells represent a powerful tool to achieve stable gene silencing. In order to interfere with DC-SIGN function, we developed shRNA-expressing lentiviral vectors capable of conditionally suppressing DC-SIGN expression. Selectivity of inhibition of human DC-SIGN and L-SIGN and chimpanzee and rhesus macaque DC-SIGN was obtained by using distinct siRNAs. Suppression of DC-SIGN expression inhibited the attachment of the gp120 envelope glycoprotein of HIV-1 to DC-SIGN transfectants, as well as transfer of HIV-1 to target cells in trans. Furthermore, shRNA-expressing lentiviral vectors were capable of efficiently suppressing DC-SIGN expression in primary human DCs. DC-SIGN-negative DCs were unable to enhance transfer of HIV-1 infectivity to T cells in trans, demonstrating an essential role for the DC-SIGN receptor in transferring infectious viral particles from DCs to T cells. The present system should have broad applications for studying the function of DC-SIGN in the pathogenesis of HIV as well as other pathogens also recognized by this receptor.     

HIV-1 replication in dendritic cells occurs through a tetraspanin-containing compartment enriched in AP-3

Dendritic cells (DC) are crucial components of the early events of HIV infection. Dendritic cells capture and internalize HIV at mucosal surfaces and efficiently transfer the virus to CD4+ T cells in trans through infectious synapses (trans-infection pathway). Alternatively, HIV-1 replicates in DC (R5-HIV-1) (cis-infection pathway). Here, we analyzed HIV trafficking in DC during the trans-infection pathway as well as the cis-infection pathway. Confocal immunofluorescence microscopy demonstrated that after capture by DC, R5-HIV-1 and HIV-1 pseudotyped with vesicular stomatitis virus protein G colocalized in a viral compartment enriched in tetraspanins including CD81, CD82 and CD9, although at different levels, indicating a role of the viral envelope in targeting to the tetraspanin-rich compartment. Replication of R5-HIV-1 in DC (cis-infection pathway) also led to the accumulation, in an envelope-independent manner, of mature viral particles in a tetraspanin-rich compartment. A fraction of the HIV-1-containing compartments appeared directly accessible from the cell surface. In sharp contrast with the trans-infection pathway, the delta-subunit of the adaptor protein 3 (AP-3) complex was enriched on the HIV-1-containing compartment during R5-HIV-1 replication in DC (cis-infection pathway). Downregulation of AP-3 delta-adaptin reduced significantly viral particle release from HIV-1-infected DC. Together, these studies demonstrate a role for AP-3 in HIV replication in a tetraspanin-rich compartment in DC and contribute to the elucidation of the trafficking pathways required for DC-T cell transfer of HIV-1 infection, a critical step during the early events of HIV infection.