Follicular T helper cells (TFH) are fundamental in orchestrating effective antibody-mediated responses critical for immunity against viral infections and effective vaccines. However, it is unclear how virus infection leads to TFH induction. We here show that dengue virus (DENV) infection of human dendritic cells (DCs) drives TFH formation via crosstalk of RIG-I-like receptor (RLR) RIG-I and MDA5 with type I Interferon (IFN) signaling. DENV infection leads to RLR-dependent IKKε activation, which phosphorylates IFNα/β receptor-induced STAT1 to drive IL-27 production via the transcriptional complex ISGF3. Inhibiting RLR activation as well as neutralizing antibodies against IL-27 prevented TFH formation. DENV-induced CXCR5+PD-1+Bcl-6+ TFH cells secreted IL-21 and activated B cells to produce IgM and IgG. Notably, RLR activation by synthetic ligands also induced IL-27 secretion and TFH polarization. These results identify an innate mechanism by which antibodies develop during viral disease and identify RLR ligands as potent adjuvants for TFH-promoting vaccination strategies.
Dengue virus is a global mosquito-transmitted pathogen that infects 400 mln people annually [1]. The majority of patients experience only mild fever, but the disease can progress to life-threatening dengue shock syndrome and dengue hemorrhagic fever. With no specific antivirals or effective vaccine available there is urgent need to advance our understanding of the human immune response against DENV to improve vaccine development and identify molecular targets for drug development. Antibodies are critical for the host immune response to control, eradicate and prevent (future) viral infections. Antibodies play a dual role in DENV pathology as neutralizing high-affinity antibodies are protective while cross-reacting antibodies can possibly enhance disease of heterologous DENV strains via antibody-dependent enhancement of infection [2]. However, it is unclear how antibodies are induced upon DENV infection. High-affinity antibodies are formed by B cells in germinal centers (GC) during somatic hypermutation [3]. TFH cells are critical for the formation and maintenance of GCs, and induce B cell proliferation and Ig isotype class switching by producing IL-21. TFH cells selectively stimulate high-affinity B cell entry into GCs to promote effective antibody-mediated responses [3–5]. Formation of TFH cells is driven by transcription factor Bcl-6, which induces IL-21 production and expression of chemokine receptor CXCR5, which is pivotal for TFH migration into GCs [6]. DCs are essential for TFH differentiation from naïve CD4+ T cells and previously we have shown that in humans IL-27 is pivotal for TFH formation while IL-6 enhances TFH formation in response to fucosylated parasitic/bacterial ligands [7]. DCs are equipped with numerous sensors including Toll-like receptors (TLRs) and RLRs that sense viral particles or viral replication products to induce innate signaling and drive TH polarization for tailored adaptive immune responses. DENV can both activate TLRs as well as RLRs, depending on the cell type, leading to cytokine secretion and type I IFN production [8–10]. TLR3 resides in endosomes while RLRs are cytoplasmic receptors. Both their activation by viral RNA leads to IFN-β induction via Tank binding protein 1 (TBK1) and transcription factor IRF3. However, RLR signaling also involves IkB kinase (IKK) -related kinase IKKε, which functions in concert with TBK1 to activate IRF3 [11,12]. In parallel with IRF3-dependent IFN-β transcription, TLR3 and RLRs activate NFκB signaling to induce cytokine expression and the combined effect of type I IFN and cytokines determines which differentiation program is initiated in naïve CD4+ T cells. However, it remains unclear how viral sensing in DCs leads to TFH development and subsequent B cell activation. Here we show that DENV-infected DCs instruct naïve CD4+ T cells to differentiate into Bcl-6+CXCR5+PD-1+ IL-21-secreting TFH cells. DENV RNA replication in both monocyte-derived and primary skin DCs triggers RLR RIG-I and MDA5 leading to IFN-β transcription and IFN-α/βR activation. Notably, RLR-induced IKKε activation modulates IFNα/βR signaling by phosphorylating STAT1. This results in the formation of transcriptional complex ISGF3 instead of STAT1 homodimers, which is pivotal for IL-27 production by DCs and TFH formation. Inhibiting RLR signaling by silencing adapter protein MAVS abrogates IL-27 production and TFH polarization by DENV-infected DCs. In addition, direct RLR activation by synthetic ligands is sufficient to induce IL-27 transcription and TFH formation.
DCs drive TH differentiation and therefore we examined DC-induced immune responses upon DENV infection. DENV efficiently infected human DCs and induced DC maturation as indicated by increased surface expression of CD83 and CD86 (S1 Fig). To investigate TFH differentiation, DENV-infected DCs were co-cultured with naïve CD4+ T cells and TFH induction was determined by measuring expression of CXCR5 and PD-1, which are both expressed by lymph node TFH cells in vivo [13,14]. Strikingly, DENV-infection of DCs induced a robust CXCR5+PD-1+ subset of differentiated TH cells (Fig 1A and 1C), which expressed high levels of TFH-specific transcription factor Bcl-6 (Fig 1B and 1D). T cell differentiation induced by DENV-infected DCs also resulted in strong secretion of IL-21, which is the main effector cytokine of TFH cells (Fig 1E). To investigate whether DENV-induced TFH cells have the capacity to activate B cells, we co-cultured DENV-differentiated TH cells with CD19+ B cells and measured antibody production. Remarkably, differentiated TH cells from DENV-infected, but not mock-treated DCs, induced secretion of both IgM and IgG by B cells (Fig 1F). Blocking DENV RNA replication and infection of DCs (S2 Fig) with DENV RNA replication inhibitor SDM25N [15] abolished the formation of IL-21-secreting CXCR5+PD-1+Bcl-6+ TFH cells (Fig 1A and 1C–1E). These data strongly indicate that DENV replication in DCs induces a TH differentiation program leading to TFH induction and B cell activation. We set out to identify the molecular mechanism in DCs essential for TFH differentiation. We investigated induction of type I IFN responses upon DENV infection. DENV induced IFN-β transcription in DCs at 18 hours post infection (h. p. i.), which increased over time and correlated with DENV RNA replication (Fig 2A). Next we measured induction of antiviral IFN stimulated genes (ISGs), which are induced by IFNα/βR signaling and indicative of functional type I IFN responses [16]. DENV-infection of DCs induced expression of ISGs MxA, APOBEC3G, ADAR1 and TRIM5α 24 h. p. i. (Fig 2B). These responses depended on DENV RNA replication as the inhibitor SDM25N abrogated the induction of IFN-β and ISGs (Fig 2C). IRF7 is crucial for the induction of IFN-α, which is required for enhancing type I IFN responses [17]. Interestingly, DENV infection induced IRF7 and IFN-α expression after IFN-β induction (Fig 2D and 2E). Both IRF7 and IFN-α expression depended on IFNα/βR signaling, while IFN-β expression was not decreased by blocking IFNα/βR antibodies at early time points (Fig 2D and 2E). IFN-β increased at 32 h. p. i. upon blocking IFNα/βR, probably because of increased DENV replication due to absence of antiviral ISGs (Fig 2E). Notably, silencing IRF7 using RNA interference abrogated IFN-α expression (Fig 2F). Thus, DENV RNA replication induces functional type I IFN responses that are initiated by IFN-β. The induction of IFNα/βR-dependent IRF7 expression subsequently drives IFN-α expression to increase and prolong type I IFN responses against DENV. Both TLRs and RLRs have been implicated in DENV sensing [8–10]. To elucidate the PRR involved in DENV-induced IFN responses in DCs, we silenced adapter molecules TRIF/MYD88 and MAVS, which are essential for TLR and RLR signaling, respectively (S3 Fig). Silencing MAVS strongly decreased IFN-β and ISG expression in DENV-infected DCs, in contrast, neither silencing of TRIF nor MYD88 affected type I IFN responses (Fig 3A, S2 Fig) even though their silencing abrogated responses to known TLR ligands (S4 Fig). Notably, silencing RIG-I and MDA5 alone or together decreased DENV-induced IFN-β as well as ISG expression (Fig 3B, S2 Fig). RLR triggering leads to activation and phosphorylation of IKKε and TBK1, which target transcription factor IRF3 for nuclear translocation to drive IFN-β expression. DENV infection induced phosphorylation of TBK1 and IKKε, which was dependent on DENV RNA replication (Fig 3C, 3D and 3E). Silencing of TBK1 and IKKε or treatment with the TBK1/IKKε inhibitor BX795 strongly decreased DENV-induced IFN-β expression and supports an important role for these kinases in the type I IFN response against DENV (Fig 3F and 3G). Moreover, DENV infection resulted in nuclear translocation of IRF3 (Fig 3H, 3I and 3J) and silencing IRF3 abrogated IFN-β expression (Fig 3K). These data show that RIG-I and MDA5 sense DENV RNA replication and induce type I IFN responses via TBK1 and IKKε-mediated IRF3 signaling. Parallel to type I IFN responses, RIG-I and MDA5 induce type III IFN responses which have been implicated in DC migration, viral suppression and modulation of T and B cell responses [18–21]. Therefore, we investigated type III IFN responses by analyzing the expression of IFN-λ genes (IFNL1-4). DENV induced the expression of IFNL1 and IFNL2 and these responses were not affected by type I IFN as blocking IFNα/βR signaling did not affect IFNL1 or IFNL2 expression (Fig 4A). Blocking IFNLR did also not impact DENV-induced IFN-α or IFN-β responses indicating that type I and type III responses operate independently (Fig 4B). IFN-λ is known to suppress viral replication and therefore we examined the effect of blocking IFNLR antibodies on DENV RNA replication. Blocking IFNLR increased DENV RNA replication but not to a similar extent as blocking IFNα/βR (Fig 4C). These data suggest that although type I and type III IFN suppress DENV replication, type I IFN is more effective than type III IFN. Next, we investigated how IFN-λ is induced by DENV. Interestingly, the replication inhibitor SDM25N abrogated DENV-induced IFNL1 and IFNL2 expression (Fig 4D). Moreover, IFNL1 and IFNL2 were induced by MAVS, RIG-I and MDA5 as silencing MAVS or RIG-I and MDA5 together strongly decreased both IFNL1 and IFNL2 expression by DENV infection (Fig 4E). These data strongly suggest that RIG-I and MDA5 triggering by DENV replication induces type III IFN responses that operate independently of type I IFN to suppress viral replication. Next, we investigated induction of cytokines involved in TFH differentiation. Mice lacking IL-27R have impaired TFH formation [22] and we have recently shown that IL-27 is crucial for TFH polarization by human DCs in response to fucosylated parasitic/bacterial ligands [23]. In addition, Activin A and IL-12 are known to be important factors to drive human TFH formation [24,25]. Therefore, we examined whether DENV infection leads to Activin A, IL-12 or IL-27 expression. IL-12 is heterodimeric protein consisting of subunit p35 and p40. Although DENV induced low levels of IL-12p35, we were unable to detect IL-12p40 expression and this resulted in a lack of IL-12p70 protein (S5A and S5B Fig). We were also unable to detect increased Activin A expression in DENV-infected cells, while LPS strongly induced both IL-12p70 and Activin A (S5A and S5B Fig). Direct stimulation of RIG-I and MDA5 with RLR ligand poly (I: C) Lyovec also did not induce IL-12p70 production or Activin A expression (S5A and S5B Fig), indicating that these factors are more associated with TLR-induced TFH formation and that RLR-mediated TFH differentiation depends on other factors. Interestingly, DENV infection of DCs induced IL-27 production, which decreased by silencing either MAVS or both RIG-I and MDA5 (Fig 5A). IL-27 is a heterodimeric cytokine consisting of subunit p28 and Epstein-Barr virus-induced gene 3 (EBI3) [26]. DENV infection induced expression of both IL-27p28 and EBI3, which was dependent on RLR signaling as well as viral replication (Fig 5B; S5C Fig). Furthermore, blocking IFNα/βR antibodies abrogated IL-27p28 expression without affecting EBI3 expression (Fig 5C), supporting differential regulation of IL-27p28 and EBI3 [27,28] and an important role for crosstalk between RLR and IFNα/βR signaling. Interestingly, IL-27p28 expression was not affected by blocking IFNLR antibodies indicating that specific IFN signaling is necessary for IL-27p28 expression (Fig 5D). Il-27p28 contains an IFN-stimulated response element (ISRE), which is induced by IFN-stimulating gene factor 3 (ISGF3), a complex of STAT1, STAT2 and IRF9 [27]. Differential signaling by IFNα/βR triggering leads to induction of either STAT1 homodimers or ISGF3 induction [29]. As ISGF3 formation is controlled by IKKε-dependent phosphorylation of STAT1 at Ser708, which prevents STAT1 homodimer formation [30] and IKKε also enhances the transactivation capacity of ISGF3 by phosphorylating STAT1 at Ser727 [23], we investigated whether ISGF3 was involved in IL-27 induction upon DENV infection. We observed STAT1 phosphorylation at Ser708 and Ser727 which was abrogated after treatment with TBK1/IKKε inhibitor BX795 (Fig 5E, 5F and 5G). We also observed nuclear translocation of IRF9 in DENV infected DCs (Fig 5H, 5I and 5J). Moreover, both IKKε and IRF9 were crucial for IL-27p28 expression since silencing of IKKε or IRF9 strongly decreased IL-27p28 expression (Fig 5K). These data show that RLR activation by DENV leads to IFN-β production and subsequent IFNα/βR triggering, and that IKKε activation modulates IFNα/βR signaling to drive IL-27 production. We next set out to investigate the importance of RLR sensing in TFH formation by DENV-infected DCs. Silencing of MAVS abrogated the formation of CXCR5+PD-1+ TFH cells by DENV-infected DCs (Fig 6A). Moreover, silencing of MAVS diminished Bcl-6 expression in T cells polarized by DENV-infected DCs (Fig 6B). These data strongly suggest that MAVS activation by DENV is pivotal for the formation of TFH cells and identifies an important role for RLR activation in TFH cells induction. To examine this, we transfected DCs with poly (I: C) or 5’pppRNA to activate RIG-I and MDA5 or RIG-I alone, respectively, and investigated TFH differentiation. Strikingly, stimulation of DCs with either poly (I: C) Lyovec or 5’pppRNA-Lyovec induced CXCR5+PD-1+ TFH formation and increased Bcl-6 expression (Fig 6E and 6F). Notably, TH cells polarized by poly (I: C) or 5’pppRNA transfected DCs activated B cells to produce IgM and IgG (Fig 6G). In contrast, LPS-stimulated DCs did neither induce TFH formation nor B cell activation (Fig 6E–6G). We next investigated the importance of IL-27 secretion by DCs for TFH formation. Interestingly, both poly (I: C) and 5’pppRNA transfected DCs produced IL-27 (Fig 6C and 6D) and neutralizing antibodies against IL-27 abrogated CXCR5+PD-1+ TFH formation (Fig 6F). Neutralizing IL-27 also diminished the capacity of TH cells polarized by poly (I: C) or 5’pppRNA transfected DCs to activate B cells to produce IgM and IgG (Fig 6G). These data strongly indicate that RLR activation, either by DENV or synthetic ligands, drives IL-27-dependent TFH polarization. DENV infection is initiated after a mosquito bite in the skin and swift immune responses against DENV depends on activation of skin DCs [31]. Human skin harbors several DC subsets including CD14+ and CD1c+ dermal DCs of which CD14+ dermal DCs are specialized in the induction of TFH cells [32]. Therefore, we isolated CD14+ and CD1c+ dermal DCs from human skin and investigated if dermal DCs mount immune responses against DENV. Interestingly, DENV induced type I IFN responses in CD1c+ as well as CD14+ dermal DCs, although the induction of type I IFN was more robust in CD14+ dermal DCs than in CD1c+ dermal DCs (Fig 7A). Notably, CD14+ dermal DCs specifically expressed IL-27p28 in response to DENV infection (Fig 7A). We next set out to investigate the importance of DENV replication in IL-27p28 expression by CD14+ dermal DCs. Remarkably, DENV RNA replication inhibitor SDM25N abrogated the induction of IL-27p28 as well as IL-27EBI3 in DENV infected CD14+ dermal DCs (Fig 7B). These data indicate that human dermal DCs mount type I IFN responses against DENV and that DENV replication is essential to trigger IL-27 expression in CD14+ dermal DCs.
TFH cells play a key role in antibody-mediated responses during viral infections or vaccination. Although numerous studies have demonstrated the importance of TFH cells in GC reactions [3–5], little is known about the factors that drive TFH differentiation from naïve CD4+ T cells upon viral infection. Here we show that DENV induces RLR-crosstalk with IFNα/βR signaling leading to IL-27 secretion, which is pivotal for the formation of IL-21-producing CXCR5+PD-1+Bcl-6+ TFH. DENV replication triggered RLR signaling leading to IKKε activation, which is essential for RLR-IFNα/βR crosstalk by phosphorylating STAT1 and inducing ISGF3 formation. Notably, direct activation of RLRs in DCs using poly (I: C) Lyovec or 5’pppRNA-Lyovec also induced IL-27 secretion, TFH polarization and IgM and IgG production by B cells. These data strongly suggest that RLRs are efficient in the induction of TFH responses via their crosstalk with IFNα/βR signaling, and links viral recognition to induction of robust antibody responses. Viral RNA is a potent pattern-associated molecular pattern that can activate numerous receptors and induce strong immune responses; both TLRs and RLRs have been implicated in DENV sensing [8–10]. DENV particles contain positive single-stranded RNA that can be directly sensed by TLR7 as shown in macrophages [8]. However, we did not find a role for TLR7 in recognizing DENV by DCs as MYD88 silencing did not affect type I IFN responses, probably because TLR7 signaling requires IRF7, which is constituently expressed in plasmacytoid DCs but minimally in other DC subsets [17,33]. Indeed, our data show that IRF7 is minimally expressed by DCS and that this transcription factor is induced by DENV infection of DCs via IFN-β. DENV replication leads to double stranded RNA intermediates, which can be sensed by TLR3 and RLRs. Studies in multiple cell lines have shown that TLR3 can be involved in IFN-β production in response to DENV infection although it is unclear how cytoplasmic RNA is transferred to endosomal TLR3 [9,34]. Our data strongly suggest that RIG-I and MDA-5 but not TLR3 are involved in sensing of DENV and subsequent induction of type I and type III IFN responses. Although DENV is known to block type I IFN responses by inhibiting RLR-MAVS interaction, TBK1 and IRF3 phosphorylation and IFNα/βR signaling via STAT2 degradation [35–38], our data suggest that the inhibition does not effectively occur in DCs. Both type I IFN and type III IFN suppressed viral replication of DENV and preventing antiviral ISG induction by IFNα/βR or IFNLR increased DENV RNA replication. Notably, the increase in DENV RNA replication resulted in an increase in IFN-β suggesting that DENV RNA and IFN-β levels are correlated. It is likely that sensing of RNA products precedes activity of de novo produced viral proteins that block RLR signaling. Recently, it was shown that Measles virus (MV) directly affects RLR activation. RIG-I and MDA5 activation is tightly regulated and requires dephosphorylation by PP1 phosphates for activation [39]. MV replication is sensed by RLRs and leads to type I IFN responses. However, MV triggers C-type lectin receptor DC-SIGN signaling leading to kinase Raf-1 activation [23]. Raf-1 subsequently induces association of inhibitor protein I-1 with PP1 to lower RIG-I and MDA5 dephosphorylation and type I IFN induction [40]. Although DENV also binds to DC-SIGN [41,42], we did not observe inhibition of RLR activation upon infection. In contrast to MV infection, which activates RLRs very rapidly [43], DENV infection is only sensed after 18 hours when innate signaling by DC-SIGN is probably not effective anymore. Therefore, RIG-I and MDA5 activation is not only important for TFH polarization and antibody production but also to limit viral replication in DCs and possible viral transmission to other cells. Indeed, our data show that RLR-dependent induction of type I IFN and type III IFN suppressed DENV. We have recently shown that IL-27 is important in the differentiation of TFH and IL-27 induction is dependent on the formation of ISGF3 [7]. Although several TLRs can induce IL-27 transcription, the levels are not sufficient to induce TFH polarization and requires IKKε activation by other receptors for STAT1 phosphorylation and ISGF3 formation [7]. RLRs are therefore unique in their ability to activate both IKKε and IFNα/βR signaling for efficient IL-27 transcription and TFH polarization. These underlying mechanisms might also apply to other viruses as Measles virus, Influenza virus, Rubella virus, HIV-1 and Hepatitis C virus activate RLRs during infection [44–47]. Indeed, our data strongly suggest the use of RLR-ligands as adjuvants for human vaccination strategies, which has been shown to be successful in animal models [48–50]. In addition to IL-27, Activin A and IL-12 have been identified as important cytokines to drive human TFH differentiation [24]. IL-12 expression is strongly induced by TLR signaling, while it is inhibited by RIG-I mediated IRF3 activation [51]. Our data also show that RLR triggering by synthetic ligands or DENV does not lead to IL-12p70 production, in contrast to strong IL-12p70 production by TLR4 activation. We obtained similar results for Activin A, suggesting that Activin A and IL-12p70 could be important for TLR-mediated TFH formation while IL-27 is crucial for RLR-mediated TFH formation. In the natural course of infection, skin DCs are the first immune cells to encounter DENV after a blood meal of an infected mosquito [31,52]. Effective control of viral propagation from the site of infection requires robust type I IFN responses to suppress viral replication. Our data show that both CD14+ and CD1c+ dermal DCs mount type I IFN responses against DENV. Interestingly, DENV specifically induced IL-27 expression in CD14+ dermal DCs, which are known to be effective inducers of TFH differentiation [32]. Our data show that the induction of IL-27 by DENV in CD14+ dermal DCs critically depends on DENV RNA replication and thereby supports a key function of cytoplasmic sensors of DENV RNA replication in the induction of TFH responses by primary human skin DCs in response to DENV infection. Our data shows that IFN-α/β induced IL-27 expression is pivotal for TFH formation by DCs while direct stimulation of naïve CD4+ cells with type I IFN-α/β is thought to inhibit TFH formation [24,53]. These studies indicate that IFN signaling, depending on the cell-type and time, can have different effects on TFH differentiation. In addition, direct IFN-α/β stimulation does not lead to IL-27 transcription without IKKε activation to modulate IFNα/βR signaling. Developing effective DENV vaccines has been hampered by the formation of non-neutralizing antibodies that have the potential to enhance disease [2,54]. A subunit vaccine based on the neutralizing epitope of DENV envelop protein could circumvent the formation of non-neutralizing antibodies [55,56]. However, subunit vaccines usually have low immunogenicity and induce only weak antibody responses. We propose that a subunit vaccine containing the neutralizing DENV epitope in combination with RLR-based adjuvants is a potent strategy to induce high levels of DENV neutralizing antibodies. In conclusion, we have identified an innate mechanism in DCs that drives TFH polarization during viral infection. Adjuvants targeting this innate mechanism have the potential to improve vaccination strategies for DENV and other pathogens.
This study was done in accordance with the ethical guidelines of the Academic Medical Center and human material was obtained in accordance with the AMC Medical Ethics Review Committee (i. e. Institutional Review Committee) according to the Medical Research Involving Human Subjects Act. Buffy coats obtained after blood donation (Sanquin) or skin tissue are not subjected to informed consent according to the Medical Research Involving Human Subjects Act and the AMC Medical Ethics Review Committee. All samples were handled anonymously. Peripheral blood monocytes were isolated from buffy coats of healthy donors (Sanquin) by Lymphoprep (Axis-Shield) gradient followed by Percoll (Amersham Biosciences) gradient steps. Monocytes were differentiated into immature DCs in the presence of 500 U/ml IL-4 and 800 U/ml GM-SCF (both Invitrogen) for 6–7 days in RPMI supplemented with 10% fetal calf serum, 10 U/ml penicillin, 10 mg/ml streptomycin (all Invitrogen) and 2 mM L-glutamine (Lonza). This study was done in accordance with the ethical guidelines of the Academic Medical Center. Dermal DCs (DDCs) were isolated from human skin tissue obtained from healthy donors after corrective breast or abdominal surgery. A dermatome (Zimmer) was used to produce 0. 3 mm skin grafts that were treated with dispase (1U/ml, Roche) for 45 min at 37°C to separate dermis and epidermis. Dermal tissue was floated on medium for 16h. Migrated cells were collected and separated based on CD14 (130-050-201, Miltenyi) and CD1c (130-090-506, Miltenyi) expression using magnetic beads according to the manufactures instruction. Isolated cells were analyzed for HLA-DR-PE/Cy7 (1: 200,560651, BD), CD11c-Alexa647 (1: 100,2108100, BioLegend), CD14-PerCP (1: 10,345786 BD) and CD1c-APC/Cy7 (1: 50,331519, BioLegend) expression on a BD Canto II (S6 Fig). CD14+ DDCs were characterized as HLA-DR+CD11c+CD14+CD1c+ and CD1c+ DDCs as HLA-DR+CD11c+CD1c+CD14-. Purity of sorted cells was over 95%. DCs were stimulated with 1 μg/ml poly (I: C) LyoVec LMW or 10 μg/ml 5’ppp-dsRNA-LyoVec (both Invivogen) unless stated otherwise. DENV replication inhibitor SDM25N (10μM, Tocris Bioscience), blocking IFNα/βR antibody or blocking IFNLR antibody (20 μg/ml, both PBL Interferon Source) were added simultaneous with DENV to DCs. DCs were transfected with 500 nM short interfering RNAs (siRNAs) using the Neon Transfection System (ThermoFisher) according to the manufacturer’s instructions. In brief, DCs were washed with PBS, resuspended in Buffer R (ThermoFisher) and divided over different siRNAs. DCs were transfected with a single pulse of 1500V for 20 ms, mixed with complete RMPI and incubated for 48h before stimulation. SMARTpool siRNA used were MAVS (M-024237-02), TRIF (M-012833-02), MYD88 (M-004769-01), RIG-I (M-012511-01), MDA5 (M-013041-00), TBK1 (M-003788-02), IKKε (M-003723-02), IRF9 (M-020858-02), IRF3 (M-006875-02), and non-targeting siRNA (D-001206-13) as control (all Dharmacon). Silencing was confirmed by real-time PCR, flow cytometry and immunoblot (S3 Fig). Naive CD4+ T cells were isolated from buffy coats of healthy blood donors (Sanquin) with human CD4+ T-cell isolation kit II (Miltenyi) by negative selection and subsequent depletion of CD45RO+ memory T cells using phycoerythrin (PE) -conjugated anti-CD45RO (80μg ml-1; R0843; Dako) and anti-PE beads (Miltenyi). B cells were isolated from buffy coats of healthy blood donors (Sanquin) with human B-cell isolation kit II (Miltenyi) by negative selection. This study was approved by the Medical Ethics Review Committee of the AMC. DCs were either silenced for indicated proteins or treated with SDM25N and stimulated for 48h as indicated. DCs were combined with allogeneic naïve CD4+ T cells (5,000 DCs/20,000 T cells) in the presence of 10 pg/ml Staphylococcus aureus enterotoxin B (Sigma). SDM25N (1 μM, Tocris Bioscience) was added to cocultures of SDM25N-treated DCs to maintain inhibition of DENV replication. Neutralizing antibodies against IL-27 (5 μg/ml, AF2526; R&D Systems) or normal goat IgG (AB-108-C; R&D Systems) as isotype control was added at the start of DC-T cell coculture. After 3 days, cells were further cultured in the presence of 10 U/ml IL-2 (Chiron). Resting T cells were restimulated with 100 ng/ml PMA and 1 μg/ml ionomycin (both Sigma) for 24h. For flow cytometry analysis of restimulated T cells, cells were stained with Alexa Fluor 647-conjugated anti-CXCR5 (1: 800; 558113; BD Pharmingen) and PerCP-Cy5. 5-conjugated α-PD-1 (1: 50; 561273; BD) before fixation in 2% para-formaldehyde for 20 min, followed by permeabilization in 50% methanol at -20°C for 45 min. Cells were stained with anti-Bcl-6 (1: 50; ab19011; Abcam), followed by incubation with PE-conjugated anti-rabbit (1: 200; 711-116-152, Jackson ImmunoResearch). Cells were analyzed on a FACS Canto II (BD Biosciences). Supernatants of restimulated T cells were harvested after 24h and IL-21 expression was analyzed by ELISA (eBioscience). T-cell dependent B-cell activation was assessed by coculturing resting differentiated T cells restimulated with 1 μg/ml anti-CD3 (1XE, Sanquin) and 2 μg/ml anti-CD28 (15E8, Sanquin) with allogeneic B cells (100,000 T cells/50,000 B cells). Supernatants were harvested after 7 days for analysis of IgM and IgG production by ELISA (eBioscience). DENV-2/16681 was added to 80% confluent C6/36 cells at an MOI of 0. 01 in RPMI medium RPMI supplemented with 2% fetal calf serum, 10 U/ml penicillin, 10 mg/ml streptomycin (all Invitrogen) and 2 mM L-glutamine (Lonza). After 5–7 days, supernatant was harvested and cleared from cellular debris by centrifugation and subsequent filtration using a 0. 2 μM filter. Supernatant was aliquoted, snap-frozen in liquid nitrogen and stored at -80°C. Viral titers were determined as described previously[57]. DCs were infected with DENV at an MOI of 1 unless stated otherwise. Infection was determined after 36-48h by flow cytometry. Cells were fixed in 4% para-formaldehyde for 15 min followed by permeabilization in PBS supplemented with 0. 1% saponin for 10 min. Cells were stained with anti-NS3 (1: 800, SAB2700181, Sigma) followed by PE-conjugated anti-rabbit (1: 200; 711-116-152, Jackson ImmunoResearch) in combination with APC-conjugated CD83 (1: 25,551073, BD Pharmingen) and FITC-conjugated CD86 (1: 25,555657, BD Pharmingen). Cells were analyzed on a FACS Canto II (BD Biosciences). TBK1, IKKε and STAT1 phosphorylation was determined by flow cytometry and immunoblot. For flow cytometry, cells were fixed in 4% para-formaldehyde for 15 min followed by permeabilization in 90% methanol at -20°C for 45 min. Cells were stained with phospho-specific antibodies against TBK1 Ser172 (1: 50,5483S, Cell Signaling), IKKε Ser172 (1: 50,06–1340, Millipore), STAT1 Ser708 (1: 100, provided by M. Gale, Jr, University of Washington School of Medicine, Seattle, WA, (Perwitasari et al. , 2011) or STAT1 Ser727 (1: 200,9177; Cell Signaling), followed by PE-conjugated anti-rabbit (1: 200; 711-116-152, Jackson ImmunoResearch). Cells were analyzed on a FACS Calibur (BD Biosciences). For immunoblot, whole cell extracts were prepared using Ripa lysis buffer (Cell Signaling Technology) and protein were resolved by SDS-PAGE and detected with anti-TBK1 Ser172 (1: 1000,5483S, Cell Signaling) anti-IKKε Ser172 (1: 1000,06–1340, Millipore), anti-STAT1 Ser708 (1: 1000, provided by M. Gale, Jr, University of Washington School of Medicine, Seattle, WA, (Perwitasari et al. , 2011) or anti-STAT1 Ser727 (1: 1000,9177; Cell Signaling). Membranes were also probed with anti-TBK1 (1: 1000,3504, Cell Signaling), anti-IKKε (1: 1000,2905, Cell Signaling) or anti-STAT1 (1: 1000,9172, Cell Signaling) as loading control. Primary antibody was detected using HRP-conjugated secondary antibody (1: 2000,21230, Pierce) mRNA was isolated using mRNA capture kit (Roche) and cDNA was synthesized with reverse transcriptase kit (Promega). PCR amplification was performed in the presence of SYBR Green in an ABI 7500 Fast PCR detection system (Applied Biosystems). Specific primers were designed using Primer Express 2. 0 (Applied Biosystems; S1 Table). Expression of target genes was normalized to GAPDH (Nt = 2Ct (GAPDH) –Ct (target) ) and set at 1 in DENV-infected DCs for each donor within one experiment. Nuclear translocation of IRF3 and IRF9 was determined by confocal microscopy, immunoblot and ELISA. For Confocal microscopy, cells were allowed to adhere to poly-l-lysine coated glass slides for 20 min at 37°C before fixation in 2% para-formaldehyde for 20 min followed by permeabilization using 0. 2% Triton for 10 min. Cells were stained with anti-IRF3 (1: 100, D83B9, Cell Signaling) or anti-IRF9 (5 μg/ml, sc-496X, Santa Cruz) and anti-DENV E protein (1: 400,3H5-1, Millipore) followed by Alexa Fluor 488-conjugated anti-mouse (1: 400, A11029, Invitrogen) and Alexa Fluor 546-conjugated anti-rabbit (1: 400, A11035, Invitrogen). Nuclei were stained using Hoechst (1: 10,000, Molecular Probes). Cells were analyzed on a Leica TCS SP8 X mounted on a Leica DMI6000 inverted microscope and data was processed using Leica LAS-X software. For immunoblot, nuclear and cytoplasmic extracts were prepared using NucBuster protein extraction kit (Novagen). Proteins were resolved by SDS-PAGE and detected by immunoblotting with anti-iRF3 (1: 1000,4302; Cell Signaling) or anti-IRF9 (1: 1000, sc-496, Santa Cruz). Membranes were also probed with anti-β-actin (1: 2500, sc-81178; Santa Cruz) to ensure equal protein loading. Detection was performed as described above. Specific increase of IRF3 and IRF9 in the nuclear fraction without increase in the cytoplasmic fraction underscores specificity of the fractionation. IRF3 and IRF9 levels in nuclear extracts was also determined using ELISA (IRF3, SEB589HU, USCN Life Sciences; IRF9, MBS921012, MyBiosource). Statistical analyses were performed using the Student’s t-test for paired observations. Statistical significance was set at P<0. 05.
Strong antibody production is critical for effective immune responses against viral infections and is a primary factor in the development of successful vaccines. However, it is unclear how virus infection leads to effective antibody responses. Dengue virus (DENV) is known to induce potent antibody production, although the underlying mechanism is poorly understood. Dendritic cells (DCs) are professional sentinels of the immune system and crucial for induction of immune responses. Here we show that DENV infection of human DCs leads to robust antibody production by inducing a specific T helper cell type (also called follicular T helper or TFH) that specializes in stimulating antibody production by B cells. Our data show that DENV replication triggers a viral detection system consisting of sensors RIG-I and MDA5, which specifically induce factors such as IL-27 that are essential for TFH induction. Our data demonstrate that this viral detection system is especially powerful to induce antibody production. Indeed, synthetic molecules that trigger this viral detection mechanism induced superior antibody production compared to other activation signals. Thus, we have identified a viral detection mechanism that leads to strong antibody production and its importance in DENV infection as well as its potential in vaccinations.
blood cells
flow cytometry
medicine and health sciences
immune cells
enzyme-linked immunoassays
immune physiology
gene regulation
immunology
microbiology
cell differentiation
developmental biology
antibodies
immunologic techniques
research and analysis methods
small interfering rnas
immune system proteins
white blood cells
animal cells
proteins
gene expression
t cells
immunoassays
viral replication
spectrophotometry
biochemistry
rna
antibody-producing cells
cytophotometry
cell biology
nucleic acids
b cells
virology
physiology
genetics
biology and life sciences
cellular types
non-coding rna
spectrum analysis techniques
RIG-I-like receptor activation by dengue virus drives follicular T helper cell formation and antibody production