Primary pDCs Research Articles

Human CD34+-derived plasmacytoid dendritic cells as surrogates for primary pDCs and potential cancer immunotherapy.

Plasmacytoid dendritic cells (pDCs) are capable of triggering broad immune responses, yet, their scarcity in blood coupled to their reduced functionality in cancer, makes their therapeutic use for in situ activation or vaccination challenging. We designed an in vitro differentiation protocol tailored for human pDCs from cord blood (CB) hematopoietic stem cells (HSCs) with StemRegenin 1 (SR-1) and GM-CSF supplementation. Next, we evaluated the identity and function of CB-pDCs compared to human primary pDCs. Furthermore, we tested the potential of CB-pDCs to support anti-tumor immune responses in co-culture with tumor explants from CRC patients. Here, we report an in vitro differentiation protocol enabling the generation of 200 pDCs per HSC and highlight the role of GM-CSF and SR-1 in CB-pDC differentiation and function. CB-pDCs exhibited a robust resemblance to primary pDCs phenotypically and functionally. Transcriptomic analysis confirmed strong homology at both, baseline and upon TLR9 or TLR7 stimulation. Further, we could confirm the potential of CB-pDCs to promote inflammation in the tumor microenvironment by eliciting cytokines associated with NK and T cell recruitment and function upon TLR7 stimulation ex vivo in patient tumor explants. This study highlights CB-pDCs as surrogates for primary pDCs to investigate their biology and for their potential use as cell therapy in cancer.

HIV-1 Nef Protein Affects Cytokine and Extracellular Vesicles Production in the GEN2.2 Plasmacytoid Dendritic Cell Line.

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon production, whose role in Human Immunodeficiency Virus (HIV) infection and pathogenesis is complex and not yet well defined. Considering the crucial role of the accessory protein Nef in HIV pathogenicity, possible alterations in intracellular signalling and extracellular vesicle (EV) release induced by exogenous Nef on uninfected pDCs have been investigated. As an experimental model system, a human plasmacytoid dendritic cell line, GEN2.2, stimulated with a myristoylated recombinant NefSF2 protein was employed. In GEN2.2 cells, Nef treatment induced the tyrosine phosphorylation of STAT-1 and STAT-2 and the production of a set of cytokines, chemokines and growth factors including IP-10, MIP-1β, MCP-1, IL-8, TNF-α and G-CSF. The released factors differed both in type and amount from those released by macrophages treated with the same viral protein. Moreover, Nef treatment slightly reduces the production of small EVs, and the protein was found associated with the small (size < 200 nm) but not the medium/large vesicles (size > 200 nm) collected from GEN2.2 cells. These results add new information on the interactions between this virulence factor and uninfected pDCs, and may provide the basis for further studies on the interactions of Nef protein with primary pDCs.

TLR1/2 orchestrate human plasmacytoid predendritic cell response to gram+ bacteria.

Gram+ infections are worldwide life-threatening diseases in which the pathological role of type I interferon (IFN) has been highlighted. Plasmacytoid predendritic cells (pDCs) produce high amounts of type I IFN following viral sensing. Despite studies suggesting that pDCs respond to bacteria, the mechanisms underlying bacterial sensing in pDCs are unknown. We show here that human primary pDCs express toll-like receptor 1 (TLR1) and 2 (TLR2) and respond to bacterial lipoproteins. We demonstrated that pDCs differentially respond to gram+ bacteria through the TLR1/2 pathway. Notably, up-regulation of costimulatory molecules and pro-inflammatory cytokines was TLR1 dependent, whereas type I IFN secretion was TLR2 dependent. Mechanistically, we demonstrated that these differences relied on diverse signaling pathways activated by TLR1/2. MAPK and NF-κB pathways were engaged by TLR1, whereas the Phosphoinositide 3-kinase (PI3K) pathway was activated by TLR2. This dichotomy was reflected in a different role of TLR2 and TLR1 in pDC priming of naïve cluster of differentiation 4+ (CD4+) T cells, and T helper (Th) cell differentiation. This work provides the rationale to explore and target pDCs in bacterial infection.

HIV-1-Mediated Suppression of IFN-α Production Is Associated with Inhibition of IRF-7 Translocation and PI3K/akt Pathway in Plasmacytoid Dendritic Cells.

Interferon-α (IFN-α) plays a vital role in combating viral infections especially in the early control after infection. However, the HIV infection has shown substantial level of suppression of IFN-α secretion during initial phase of infection. The reasons behind this impairment are still obscure. As plasmacytoid dendritic cells (pDCs) are the major producers of this cytokine, the mechanisms of HIV-1-mediated suppression of IFN-α production by pDCs using the primary pDCs were explored. The nuclear translocation of the interferon regulatory factor (IRF)-7, a transcription factor for IFN-α genes, is essential for the initiation of IFN-α production in pDCs. The HIV-1-exposed pDCs did not show the translocation of IRF-7 into the nucleus in our experiments. Furthermore, it was also observed that HIV-1 inhibited AKT phosphorylation of PI3K/akt pathway in pDCs, an important step for IRF-7 translocation to nucleus. HIV-1-induced inhibition of AKT phosphorylation and IRF-7 translocation was evident even in the presence of Toll-like receptor-7 agonist stimulation and correlated with IFN-α suppression. The findings suggest that HIV-1 may alter AKT phosphorylation to inhibit the translocation of IRF-7 into pDC nucleus, leading to IFN-α suppression, and this may be the reason for IFN-α abrogation observed in recently infected HIV patients. Understanding of interactions between HIV-1 and signaling pathways leading to IFN-α secretion may provide targets for immune intervention.

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile.

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells.

Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, α/β/ω) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

Dual Role of the Tyrosine Kinase Syk in Regulation of Toll-Like Receptor Signaling in Plasmacytoid Dendritic Cells.

Crosslinking of regulatory immunoreceptors (RR), such as BDCA-2 (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses production of type-I interferon (IFN)-α/β and other cytokines in response to Toll-like receptor (TLR) 7/9 ligands. This cytokine-inhibitory pathway is mediated by spleen tyrosine kinase (Syk) associated with the ITAM-containing adapter of RR. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via RR, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-α, TNF-α and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or RR signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and RR signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-α production triggered via the RR pathway, including that induced by hepatitis B and C viruses. Thus, pharmacological targeting of Syk partially restored the main pDC function—IFN-α production. Opposing roles of Syk in TLR7/9 and RR pathways may regulate the innate immune response to weaken inflammation reaction.

S1PR4 Signaling Attenuates ILT 7 Internalization To Limit IFN-α Production by Human Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type I IFN in response to TLR7/9 ligands. This conveys antiviral effects, activates other immune cells (NK cells, conventional DCs, B, and T cells), and causes the induction and expansion of a strong inflammatory response. pDCs are key players in various type I IFN-driven autoimmune diseases such as systemic lupus erythematosus or psoriasis, but pDCs are also involved in (anti-)tumor immunity. The sphingolipid sphingosine-1-phosphate (S1P) signals through five G-protein-coupled receptors (S1PR1-5) to regulate, among other activities, immune cell migration and activation. The present study shows that S1P stimulation of human, primary pDCs substantially decreases IFN-α production after TLR7/9 activation with different types of CpG oligodeoxynucleotides or tick-borne encephalitis vaccine, which occurred in an S1PR4-dependent manner. Mechanistically, S1PR4 activation preserves the surface expression of the human pDC-specific inhibitory receptor Ig-like transcript 7. We provide novel information that Ig-like transcript 7 is rapidly internalized upon receptor-mediated endocytosis of TLR7/9 ligands to allow high IFN-α production. This is antagonized by S1PR4 signaling, thus decreasing TLR-induced IFN-α secretion. At a functional level, attenuated IFN-α production failed to alter Ag-driven T cell proliferation in pDC-dependent T cell activation assays, but shifted cytokine production of T cells from a Th1 (IFN-γ) to a regulatory (IL-10) profile. In conclusion, S1PR4 agonists block human pDC activation and may therefore be a promising tool to restrict pathogenic IFN-α production.

Sex Differences in Plasmacytoid Dendritic Cell Levels of IRF5 Drive Higher IFN-α Production in Women.

Increased IFN-α production contributes to the pathogenesis of infectious and autoimmune diseases. Plasmacytoid dendritic cells (pDCs) from females produce more IFN-α upon TLR7 stimulation than pDCs from males, yet the mechanisms underlying this difference remain unclear. In this article, we show that basal levels of IFN regulatory factor (IRF) 5 in pDCs were significantly higher in females compared with males and positively correlated with the percentage of IFN-α-secreting pDCs. Delivery of recombinant IRF5 protein into human primary pDCs increased TLR7-mediated IFN-α secretion. In mice, genetic ablation of the estrogen receptor 1 (Esr1) gene in the hematopoietic compartment or DC lineage reduced Irf5 mRNA expression in pDCs and IFN-α production. IRF5 mRNA levels furthermore correlated with ESR1 mRNA levels in human pDCs, consistent with IRF5 regulation at the transcriptional level by ESR1. Taken together, these data demonstrate a critical mechanism by which sex differences in basal pDC IRF5 expression lead to higher IFN-α production upon TLR7 stimulation in females and provide novel targets for the modulation of immune responses and inflammation.

Oxidative modification enhances the immunostimulatory effects of extracellular mitochondrial DNA on plasmacytoid dendritic cells

Inflammation is associated with oxidative stress and characterized by elevated levels of damage-associated molecular pattern (DAMP) molecules released from injured or even living cells into the surrounding microenvironment. One of these endogenous danger signals is the extracellular mitochondrial DNA (mtDNA) containing evolutionary conserved unmethylated CpG repeats. Increased levels of reactive oxygen species (ROS) generated by recruited inflammatory cells modify mtDNA oxidatively, resulting primarily in accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) lesions. In this study, we examined the impact of native and oxidatively modified mtDNAs on the phenotypic and functional properties of plasmacytoid dendritic cells (pDCs), which possess a fundamental role in the regulation of inflammation and T cell immunity. Treatment of human primary pDCs with native mtDNA up-regulated the expression of a costimulatory molecule (CD86), a specific maturation marker (CD83), and a main antigen-presenting molecule (HLA-DQ) on the cell surface, as well as increased TNF-α and IL-8 production from the cells. These effects were more apparent when pDCs were exposed to oxidatively modified mtDNA. Neither native nor oxidized mtDNA molecules were able to induce interferon (IFN)-α secretion from pDCs unless they formed a complex with human cathelicidin LL-37, an antimicrobial peptide. Interestingly, simultaneous administration of a Toll-like receptor (TLR)9 antagonist abrogated the effects of both native and oxidized mtDNAs on human pDCs. In a murine model, oxidized mtDNA also proved a more potent activator of pDCs compared to the native form, except for induction of IFN-α production. Collectively, we demonstrate here for the first time that elevated levels of 8-oxoG bases in the extracellular mtDNA induced by oxidative stress increase the immunostimulatory capacity of mtDNA on pDCs.

TLR ligands upregulate RIG-I expression in human plasmacytoid dendritic cells in a type I IFN-independent manner.

Plasmacytoid dendritic cells (pDCs) are professional type I interferon (IFN)-producing cells that play an essential role in antiviral immunity. In many cell types, detection of intracellular pathogens is mostly dependent on endosomal Toll-like receptors (TLRs) and cytosolic sensors, such as retinoic acid-inducible gene I (RIG-I). However, the possible interplay between these two systems has not yet been elucidated. Here we aimed to study the collaboration of endosomal TLRs and RIG-I in primary human pDCs. We found that under steady-state conditions, pDCs express RIG-I at very low level, but the expression of this receptor is rapidly and dramatically upregulated upon stimulation by the TLR7 ligand imiquimod or the TLR9 ligand type A CpG. We also demonstrated that pDCs are able to sense and respond to 5'-triphosphate double-stranded RNA (5'-ppp-dsRNA) only following activation by endosomal TLRs. Experiments on primary pDCs with functionally blocked IFN-α/β receptor 1 (IFNAR1) and those on human pDC leukemia (pDC-L) cells defective in type I IFN secretion indicated that the upregulation of RIG-I expression in pDCs upon stimulation by endosomal TLR occurs in a type I IFN-independent manner. Selective phosphorylation of signal transducer and activator of transcription 1 (STAT1) on tyrosine 701 could be identified as an early signaling event in this process. Our results show that in contrast to many other cell types, where RIG-I expression is induced by type I IFN, in pDCs a disparate mechanism is responsible for the upregulation of RIG-I. Our findings also indicate that along with autophagy, an additional mechanism is operating in pDCs to promote the detection of replicating viruses.

Natural mannosylation of HIV-1 gp120 imposes no immunoregulatory effects in primary human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity, and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs, but no receptors for high-mannose have so far been reported on human pDCs. Here we show that upon activation with HIV-1 or by a synthetic compound triggering the same receptor in human pDCs as single-stranded RNA, human pDCs upregulate the mannose receptor (MR, CD206). To examine the functional outcome of this upregulation, inactivated intact or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha, viability, and upregulation of several pDC-activation markers: CD40, CD86, HLA-DR, CCR7, and PD-L1. The level of activation negatively correlated with degree of mannosylation, however, subsequent reduction in the original mannosylation level had no effect on the pDC phenotype. Furthermore, two of the infectious HIV-1 strains induced profound necrosis in pDCs, also in a mannose-independent manner. We therefore conclude that natural mannosylation of HIV-1 is not involved in HIV-1-mediated immune suppression of pDCs.

HIV cell-to-cell spread and innate immune responses

Our research is aimed at understanding the interplay between viruses and the immune system. HIV cell-tocell transmission is a major mechanism of viral spread. We are studying how antiviral molecules, such as broadly neutralizing antibodies and restriction factors, known to inhibit infection with cell-free virions, interfere with HIV-1 cell-to-cell transmission. Moreover, we previously showed that HIV-1 infected lymphocytes are more potent inducers of type-l IFN than free virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7. In myeloid DCs, and other cells that lack TLR7, recognition is independent of TLR7, and occurs in a large part through a cytoplasmic pathway that we are currently analyzing. Finally, we are examining how HIV-2 spreads from cell-to-cell and triggers type-l IFN in various primary cell types. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

MiR-29b and miR-29c Are Involved in Toll-Like Receptor Control of Glucocorticoid-Induced Apoptosis in Human Plasmacytoid Dendritic Cells

Glucocorticoids (GCs) are frequently used to treat many of the acute disease manifestations associated with inflammatory and autoimmune disorders. However, Toll-like receptor (TLR) pathway-activated plasmacytoid dendritic cells (pDCs) are resistant to GC-induced apoptosis, which leads to the inefficiency of GCs in the treatment of type I interferon-related autoimmune diseases, such as systemic lupus erythematosus (SLE). Therefore, compounds promoting pDC apoptosis may be helpful for improving the efficacy of GCs. In this study, we performed screening to identify microRNAs (miRNAs) involved in TLR-inhibited GC-induced pDC apoptosis and found an array of miRNAs that may regulate pDC apoptosis. Among those demonstrating altered expression, 6 miRNAs were inhibited in TLR-activated pDCs. Bioinformatics analysis and functional studies indicated that miR-29b and miR-29c were 2 key miRNAs involved in TLR-inhibited GC-induced pDC apoptosis. Furthermore, both of these miRNAs promoted pDC apoptosis by directly targeting Mcl-1 and Bcl-2 in human primary pDCs. Our findings provide new targets that could improve the efficacy of GCs for the treatment of SLE.

CD2AP/SHIP1 Complex Positively Regulates Plasmacytoid Dendritic Cell Receptor Signaling by Inhibiting the E3 Ubiquitin Ligase Cbl

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.

Phospholipid Scramblase 1 regulates Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells

Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells (pDCs) and triggers MyD88-dependent type I interferon (IFN) responses. To better understand TLR9 biology in pDCs, we established a yeast two-hybrid library for the identification of TLR9-interacting proteins. Here, we report that an IFN-inducible protein, phospholipid scramblase 1 (PLSCR1), interacts with TLR9 in pDCs. Knockdown of PLSCR1 expression by siRNA in human pDC cell line led to a 60-70% reduction of IFN-α responses following CpG-ODN (oligodeoxynucleotide) stimulation. Primary pDCs from PLSCR1-deficient mice produced lower amount of type 1 IFN than pDCs from the wild-type mice in response to CpG-ODN, herpes simplex virus and influenza A virus. Following CpG-A stimulation, there were much lower amounts of TLR9 in the early endosomes together with CpG-A in pDCs from PLSCR1-deficient mice. Our study demonstrates that PLSCR1 is a TLR9-interacting protein that plays an important role in pDC's type 1 IFN responses by regulating TLR9 trafficking to the endosomal compartment.

The double-stranded RNA bluetongue virus induces type I interferon in plasmacytoid dendritic cells via a MYD88-dependent TLR7/8-independent signaling pathway.

Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/β) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/β production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/β induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/β in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/β. BTV replication in pDCs was not mandatory for IFN-α/β production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/β required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/β induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/β in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses.

Novel insights into innate sensing of HIV-infected cells

Our research is aimed at understanding the interplay between viruses and the immune system. We are analyzing how HIV infection triggers production of type-l interferon (IFN) and other cytokines in various cell types. Cell-free HIV-1 virions are considered to be poor stimulators of IFN production. We examined how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs), by monocyte-derived DCs (MDDCs), and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate viral sensing. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. We are also currently analyzing the role of SAMHD1, a recently identified anti-HIV restriction factor, in the activation of MDDCs by HIV. Altogether, our results indicate that detection of HIV-infected lymphocytes occurs at different levels, through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

Continuous long-term growth of plasmacytoid dendritic cells following in vitro infection with HTLV-1

Plasmacytoid dendritic cells (pDCs) isolated from HTLV-1-infected individuals express viral proteins and can infect CD4+ T cells in vitro [1], and the proviral load in pDCs parallels the PBMC proviral load in infected individuals [2], suggesting a role for these cells in HTLV-1 transmission and persistence. We recently generated immortalized cell lines from primary pDCs infected with HTLV-1. Peripheral blood pDCs can survive in ex vivo culture for a limited time in the presence of IL-3. Following infection with cell-free virus, pDC were cultured with activated CD4+ T cells, and separated by immunomagnetic beads. Cells capable of continuously proliferating in the absence of IL-3 were isolated and characterized. FACS analysis revealed that these cells are phenotypically similar to pDCs (CD123+, BDCA2+, CMKLR1+, CD3-, CD19-, CD14-). Preliminary studies suggest that these cells spontaneously produce cytokines and chemokines expressed by pDCs. These pDC-like cells produce high levels of HTLV-1, as determined by ELISA analyses of culture supernatants and transmission EM and, like freshly isolated pDCs, can efficiently transmit HTLV-1 to T cells. As previously shown for HTLV-1-infected T cells [3], expression of HTLV-1 in these pDC-like cells is markedly decreased following exposure to type 1 interferons. Studies to characterize the viral integration sites and to examine expression levels of proteins previously shown to be altered in HTLV-1-transformed T cells are currently in progress. The in vitro generation of immortalized HTLV-1-infected pDC-like cells capable to transmitting the virus to T cells support the notion that pDCs could be an in vivo reservoir for HTLV-1.

Innate Sensing of HIV-Infected Cells

Cell-free HIV-1 virions are poor stimulators of type I interferon (IFN) production. We examined here how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs) and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. Therefore, detection of HIV-infected lymphocytes occurs through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.