CD8 T Cell Priming Type I IFN Response Needed for Efficient

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Conventional but Not Plasmacytoid Dendritic Cells Foster the Systemic Virus −Induced Type I IFN Response Needed for Efficient CD8 T Cell Priming This information is current as of September 29, 2021. Sandra Hervas-Stubbs, Jose-Ignacio Riezu-Boj, Uxua Mancheño, Paloma Rueda, Lissette Lopez, Diego Alignani, Estefanía Rodríguez-García, Nathalie Thieblemont and Claude Leclerc

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Conventional but Not Plasmacytoid Dendritic Cells Foster the Systemic Virus–Induced Type I IFN Response Needed for Efﬁcient CD8 T Cell Priming

Sandra Hervas-Stubbs,*,1 Jose-Ignacio Riezu-Boj,* Uxua Manchenõ,* Paloma Rueda,† Lissette Lopez,† Diego Alignani,* Estefanıá Rodrı´guez-Garcıá,* Nathalie Thieblemont,‡ and Claude Leclercx,{,1

Plasmacytoid dendritic cells (pDCs) are considered to be the principal type-I IFN (IFN-I) source in response to viruses, whereas the contribution of conventional DCs (cDCs) has been underestimated because, on a per-cell basis, they are not considered professional IFN-I–producing cells. We have investigated their respective roles in the IFN-I response required for CTL activation. Using a nonreplicative virus, baculovirus, we show that despite the high IFN-I–producing abilities of pDCs, in vivo cDCs but not pDCs Downloaded from are the pivotal IFN-I producers upon viral injection, as demonstrated by selective pDC or cDC depletion. The pathway involved in the virus-triggered IFN-I response is dependent on TLR9/MyD88 in pDCs and on stimulator of IFN genes (STING) in cDCs. Importantly, STING is the key molecule for the systemic baculovirus-induced IFN-I response required for CTL priming. The supremacy of cDCs over pDCs in fostering the IFN-I response required for CTL activation was also veriﬁed in the lymphocytic choriomeningitis virus model, in which IFN-b promoter stimulator 1 plays the role of STING. However, when the TLR- independent virus-triggered IFN-I production is impaired, the pDC-induced IFNs-I have a primary impact on CTL activation, http://www.jimmunol.org/ as shown by the detrimental effect of pDC depletion and IFN-I signaling blockade on the residual lymphocytic choriomeningitis virus–triggered CTL response detected in IFN-b promoter stimulator 12/2 mice. Our ﬁndings reveal that cDCs play a major role in the TLR-independent virus-triggered IFN-I production required for CTL priming, whereas pDC-induced IFNs-I are dispensable but become relevant when the TLR-independent IFN-I response is impaired. The Journal of Immunology, 2014, 193: 1151–1161.

endritic cells (DCs) are considered to be the only APCs maturation signals to DCs and by modulating the degree of Ag that are able to prime naive CD8 T cells owing to their presentation and costimulation (5). Furthermore, T cell–intrinsic

D unique ability to process and present Ag, to express IFN-I signaling was reported to substitute for T cell help to CTLs by guest on September 29, 2021 costimulatory molecules, and to release soluble mediators, such as (6). Although not considered specifically to be signal 3 cytokines, type I IFNs (IFNs-I) and other inflammatory cytokines, which are other cytokines, such as the common cytokine receptor g-chain crucial for the efficient triggering of the CTL response (1, 2). With family (7) and IFN-g (8), also have a profound effect on the CTL these three functions, DCs link the innate and adaptive immune response. systems. The two main DC subsets, conventional DCs (cDCs) and Recent studies indicate that IFNs-I (mainly IFN-a and IFN-b) plasmacytoid DCs (pDCs) (9), are widely considered to have and IL-12 act directly on naive CD8 T cells, responding to Ag and highly specialized functions in the induction of a CTL response. costimulation by favoring their priming (3, 4). Stimulation of Thus, whereas cDCs are considered to be the main APCs, pDCs naive CD8 T cells in the absence of these signal 3 cytokines leads are thought to be the principal sources of IFN-I production in to proliferation but results in poor survival and the failure to de- responses to viruses (10). However, these two DC functions are velop optimal effector and memory functions (4). IFNs-I also not so strictly compartmentalized. Indeed, although less efficient promote CTL responses in an indirect manner by delivering than cDCs, TLR- or virus-licensed pDCs are able to cross-present

*Division of Immunology and Immunotherapy, Center for Applied Medical Re- plied Medical Research, University of Navarra, Avenida de Pio XII, 55 Pamplona search, University of Navarra, Pamplona 31008, Spain; †Inmunologıá y Gene´tica 31008, Spain (S.H.-S.) or Institut Pasteur, Unite´ de Re´gulation Immunitaire et Aplicada, S.A., Madrid 28037, Spain; ‡Centre National de la Recherche Scientifique, Vaccinologie, 25 Rue du Docteur Roux, 75724 Paris, Cedex 15, France (C.L.). Unite´ Mixte de Recherche 8147, Universite´ Rene´ Descartes Paris V, Hoˆpital Necker, E-mail addresses: [email protected] (S.H.-S.) or [email protected] (C.L.) Paris F-75015, France; xInstitut Pasteur, Unite´ de Re´gulation Immunitaire et Vacci- { The online version of this article contains supplemental material. nologie, Paris F-75015, France; and INSERM, Unite´ 1041, Paris F-75015, France Abbreviations used in this article: BEI, binary ethylenimine; BM, bone marrow; 1S.H.-S. and C.L. share senior authorship. BOVAp, beads linked to the OVA257–264 peptide; BV, baculovirus; cDC, conventional Received for publication June 4, 2013. Accepted for publication May 27, 2014. DC; DC, dendritic cell; DT, diphteria toxin; DTR, diphtheria toxin receptor; IFNAR, IFN-a/b receptor; IFN-I, type I IFN; IPS-1, IFN-b promoter stimulator 1; LCMV, This work was supported by grants from the Ligue Nationale Contre le Cancer lymphocytic choriomeningitis virus; LCMVArm, LCMV Armstrong strain; MDA-5, (Equipe Labelliseé 2014), the Institut National du Cancer/Cance´ropole Ile de France, melanoma differentiation-associated protein 5; MIG, monokine induced by IFN-g; the Banque Priveé Europeénne, the European Union’s Seventh Framework Pro- ODN, oligonucleotide; OVAp, OVA peptide; PAMP, pathogen-associated mo- gramme (FP7/2007-2013) under Grant 280873 (Advanced Immunization Tech- 257–264 lecular pattern; pDC, plasmacytoid DC; STING, stimulator of IFN genes; TRIF, Toll/ nologies), Fondo de Investigacioń Sanitaria Grant PI11/02327, and by the Unioń IL-1R domain–containing adapter inducing IFN-b; WT, wild-type. Temporal de Empresas project Centro de InvestigaciońMe´dica Aplicada. S.H.-S. was supported by the Asociacioń Espanõla Contra el Cancer and by Ministerio de This article is distributed under The American Association of Immunologists, Inc., Educacioń y Ciencia Grant RYC-2007-00928. Reuse Terms and Conditions for Author Choice articles.

Address correspondence and reprint requests to Dr. Sandra Hervas-Stubbs or Ó Dr. Claude Leclerc, Division of Immunology and Immunotherapy, Center for Ap- Copyright 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301440 1152 CELLULAR SOURCES OF IFNs-I FOR CTL PRIMING particulate Ags and to cross-prime naive CD8 T cells (11). mice were obtained from The Jackson Laboratory. Animals were kept Moreover, there is growing evidence that pDCs are dispensable for under specific pathogen-free conditions. Experiments involving animals IFN-I responses to certain viruses, suggesting that other cells must were conducted according to institutional guidelines. be involved in the production of IFNs-I. In this regard, it has BV and LCMV become increasingly clear that pDCs are essential for the production of IFNs-I only during the initial stages of murine CMV Purified Autographa californica nuclear polyhedrosis virus (referred to as BV) was produced by Agate Bioservices (Bagard, France) as described and vesicular stomatitis virus (VSV) infection (12–14). Further- previously (29). Virus stock was free of endotoxin (,0.01 endotoxin units/ more, pDCs only efficiently control the IFN-I response when low- ml) (Limulus amebocyte lysate test, QCL-1000; BioWhittaker/Cambrex). to-intermediate doses of murine CMV are administered but are BV particles (109 PFU/ml) were inactivated either by exposure to UV light 4 2 insufficient at high viral loads (13). Additionally, mice treated (2 3 10 mJ/cm ) using a UV Crosslinker (Amersham Life Science) or by with the 120G8 mAb for pDC depletion had the same IFN-a treatment with binary ethylenimine (BEI) or Triton X-100 as previously described (29). We detected no infectious BV after UV light or BEI levels in response to infection with the influenza virus strain X31 treatment and only a residual titer of 7 3 103 PFU/ml after Triton X-100 as did untreated mice (15). Although it has been reported that treatment (29). For Benzonase treatment, BV was incubated with Benzo- depletion of pDCs in respiratory syncytial virus infection nase (90 U/ml) (Novagen) and MgCl2 (2 mM) (2 h, 37˚C). Benzonase was decreases viral clearance (16), other studies suggest that IFN-I then inactivated with 150 mM NaCl. DNA from BV was isolated by phenol-chloroform-isoamyl alcohol extraction from purified virions as production by pDCs in respiratory syncytial virus infection may described previously (29). The precipitated DNA was resuspended in not be a critical factor (17). Moreover, Ikaros L/L mice, which sterile endotoxin-free water. No protein or chromosomal DNA from the lack peripheral pDCs but harbor normal numbers of cDCs, mount insect cells was detected. LCMV Armstrong (LCMVArm) strain was efficient B cell– and T cell–specific responses against the influ- grown, stored, and quantified according to published methods (30). Downloaded from enza virus (18). Other studies have shown that conditional deletion DC purification or Ab-mediated depletion of pDCs does not alter the induction of an antiviral CTL response (13, 19). For the purification of splenic cDCs and pDCs, spleens were harvested and The role of cDCs as a source of IFNs-I against viruses could treated for 15 min with 400 U/ml collagenase D and 50 mg/ml DNase I be underestimated simply because, on a per-cell basis, they are (Boehringer Mannheim, Mannheim, Germany). The cell suspension was enriched in pDCs and cDCs by depleting splenocytes of B, T, and NK cells nonprofessional IFN-I–producing cells. However, cDCs are also with anti-CD19, anti-CD90, and anti-DX5 mAb–coated magnetic beads http://www.jimmunol.org/ able to produce appreciable levels of IFN-a under certain con- (Miltenyi Biotec, Bergisch Gladbach, Germany) on an autoMACS (Mil- ditions (20, 21), and because they exceed pDCs in number (22), tenyi Biotec) using the DepleteS program. The DC-enriched fraction was these nonprofessional IFN-a/b–producing cells may also play an further purified on a FACSAria cell sorter (BD Biosciences, San Diego, CA). Briefly, cells were stained with anti-CD16/32 (Fc Block), anti–mouse important role in regulating the virus-triggered IFN-I response CD19-PerCP-Cy5.5 (6D5), CD3-PerCP-Cy5.5 (145-2C11), NK1.1-PerCP- necessary for CTL priming. Therefore, the precise cellular source Cy5.5 (PK-136) (or pan-NK mAb [DX5] for 129sv cells), F4-80–PerCP- of virus-induced IFN-I production leading to CTL activation Cy5.5 (BM8), CD11c-PE (N418), and B220-allophycocyanin (RA3-6B2) + + + + + remains poorly understood. Given the importance of DCs for the mAbs. Contaminating CD19 , CD3 , F4-80 , and NK1.1 (or DX5 ) cells were gated out and pDCs and cDCs were sorted on the basis of CD11c and priming of naive CD8 T cells, it is critical to define the relative int + high 2 B220 expression (pDCs, CD11c B220 ; cDCs, CD11c B220 ) from by guest on September 29, 2021 contributions of pDCs and cDCs to the inflammatory milieu DC-enriched splenocytes. necessary for CTL activation. To explore this we have used both replicative (lymphocytic In vitro DC stimulation choriomeningitis virus [LCMV]) and nonreplicative (baculovirus Purified splenic cDCs and pDCs (105 cells/well) were cultured in complete [BV]) viruses as danger signal sources. Using the BV model we RPMI medium in the presence of 107 PFU/ml BV, either untreated or show that on a per-cell basis, pDCs are higher IFN-I producers than treated with UV light, BEI, Triton X-100, or Benzonase. As control, DCs are cDCs upon both in vivo and in vitro virus stimulation. However, were also stimulated with a volume of Benzonase (90 U/ml) equivalent to in vivo, the nonprofessional IFN-I–producing cDCs are the main the volume of Benzonase-treated BV used. In other cases, DCs were stimulated with 1:10 diluted supernatant from Sf9 cell culture (72 h) source of IFN-I in response to both BV and LCMV, as shown by infected or uninfected with BV. The BV titer in the supernatant from BV- selective depletion of pDCs or cDCs. Thus, caution should be infected Sf9 cells was 107 PFU/ml. DCs were also stimulated with BV- taken when extrapolating IFN-I–producing abilities to infer the DNA (25 mg/ml), CpGA-2216 (25 mg/ml) (Genset), or R848 (1 mg/ml) impact of a DC subset on the in vivo virus-triggered IFN-I re- (InvivoGen). Supernatants were harvested after 36 h of culture. sponse. Importantly, we show that by sensing the virus through Immunizations TLR-independent pathways, cDCs foster the IFN-I response re- 9 quired for efficient CTL priming, whereas pDCs were not re- In the BV model, mice were i.v. immunized with 10 synthetic latex beads quired. However, in case of an impaired virus-triggered systemic (1 mm diameter) (Polysciences) covalently linked to the OVA257-264 synthetic peptide (BOVAp; Neosystem, Strasbourg, France), as previously IFN-I production, the pDC-induced IFN-I response has a major described (31), either alone or in combination with BV (106 PFU). We role on CTL priming. These results thus highlight the role played chose BOVAp because the CTL response induced with this immunogen is by different cellular sources of IFN-I on CTL priming and could fully dependent on the adjuvant signals provided by live BV or TLR ligands (29, 32). In the LCMV model, mice were first injected (i.v.) with lead to improvement in the development for IFN-a–based thera- 5 LCMVArm (6 3 10 PFU) and 15 h later with 500 mg soluble OVA257–264 pies for viral and malignant diseases. synthetic peptide (s.c.).

Materials and Methods mAb treatments Mice For pDC in vivo depletion, C57BL/6J mice received two i.v. injections (24-h Female C57BL/6J and 129Sv mice were from Charles River Laborato- interval) of 500 mg/injection of functional-grade pure anti–mPDCA-1 ries (L’Arbresle, France). C57BL/6J mice lacking TLR9 (23), MyD88 mAbs (Miltenyi Biotec) or control rat IgG2b (eBioscience). The percent- (24), and IFN-b promoter stimulator 1 (IPS-1) (25) were obtained from age of depletion was calculated as [(no. cells before depletion 2 no. cells Dr. S. Akira. Toll/IL-1R domain–containing adapter inducing IFN-b after depletion)/cells before depletion] 3 100. To block IFN-I signaling, (Trif)2/2 mice were obtained from Dr. S. Akira and backcrossed onto mice were treated with 0.5 mg anti–IFN-a/b receptor (IFNAR1) Ab i.p. a C57BL/6J background (11 backcrosses) (26). Stimulator of IFN genes (clone MAR1-5A3) (Leincon Technologies) or with a mouse IgG1 isotype (STING)2/2 (C57BL/6J-Tmem173gt/J) (27) and CD11c–diphteria toxin control (clone MOPC21) (Bio-X-Cell) on days 21 and 0 of BV injection receptor (DTR) (B6.FVB-Tg(Itgax-DTR/GFP)57Lan/J) (28) transgenic and on days 21, 0, 1, and 2 of LCMVArm infection. The Journal of Immunology 1153

Generation of CD11c-DTR → wild-type chimeric mice and CTLs, whereas Benzonase treatment, which digests soluble but cDC depletion not virus-packaged ODNs, does not (29). These data suggest that CD11c-DTR → wild-type (WT) bone marrow (BM) chimeric mice were ODNs enclosed in the virion, presumably the viral DNA, are the generated by irradiation of recipient mice (C57BL/6J) with a single lethal pathogen-associated molecular pattern (PAMP) molecules re- dose of 700 cGy. Recipient mice were then reconstituted with 5 3 106 sponsible for the immunostimulatory properties of BV. donor BM cells from CD11c-DTR mice. Chimeric mice were kept on To determine the role of the TLR9/MyD88 pathway in the in vivo antibiotic-containing drinking water for 10 d and allowed to reconstitute → production of inflammatory cytokines triggered by BV, we injected for 8 wk before use. For systemic cDC depletion, CD11c-DTR WT 2/2 2/2 chimeric mice were i.p. injected with 100 mg DT (Calbiochem) 24 and WT, TLR9 , and MyD88 mice with BV. Interestingly, the 96 h before BV injection or 24 and 72 h before LCMVArm injection. production of IL-12p40, MIG, and especially IFN-g was partially dependent on the TLR9/MyD88 pathway, in contrast to IFN-a/b Cytokine detection assays production, which was totally independent of this pathway IFN-a,IFN-b, and monokine induced by IFN-g (MIG) levels were (Fig. 1A). The BV-induced cytokine response was fully indepen- detected using ELISA kits (IFN-a and IFN-b from PBL Biomedical dent of Trif (Supplemental Fig. 1A). Laboratories, MIG from R&D Systems). IL-12p40, IL-12p70, and IFN-g Cytosolic DNA sensors, such as DNA-dependent activator of were measured by in-house ELISA (33). IFN regulatory factor-1 and IFN-g–inducible protein 16, which Flow cytometry analysis signal through STING, have been also involved in the sensing of Cell staining was performed with fluorochrome-conjugated mAbs in the presence of purified anti-CD16/32 mAb. Appropriate isotype controls were used to verify staining specificity. mAbs used in this study are: mAb against Downloaded from CD19 (6D5), CD3 (145-2C11), NK1.1 (PK-136) (or pan-NK mAb [DX5] for 129sv cells), F4-80 (BM8), CD11c (N418), B220 (RA3-6B2), CD11b (M1/70), GR1 (RB6-8C5), I-Ab (AF6-120.1), Ly6G (1A8), and CD8 (53-6- + 7). The number of OVA257–264-specific CTLs (OVATetr ) was determined b by staining spleen cells first with H-2K –OVA257–264-tetramer-PE (Beckman Coulter) and then with anti–CD3-allophycocyanin and anti–

CD8-FITC mAbs. Cells were acquired on either a FACSCalibur or http://www.jimmunol.org/ FACSCanto II ﬂow cytometer and analyzed using CellQuest software (BD Biosciences) or FlowJo (Tree Star, San Carlos, CA).

Real-time RT-PCR Total RNA was extracted using nucleic acid puriﬁcation lysis solution and a semiautomated ABI Prism 6100 Nucleic Acid PrepStation system (Ap- plied Biosystems). Total RNA was treated with DNase prior to reverse transcriptase with Moloney murine leukemia virus in the presence of RNaseOUT (all from Invitrogen). Real-time RT-PCR was performed using

the CFX96 real-time system, iQ SYBR Green mix (Bio-Rad), and specific by guest on September 29, 2021 primers for each gene: mouse IFN-a (sense, 59-TCTYTCYTGYCTGAA- GGAC-39, antisense, 59-CACAGRGGCTGTGTTTCTTC-39) (degenerate primers for all mouse IFN-a subtypes) (34), mouse IFN-b (sense, 59-ATG- AGTGGTGGTTGCAGGC-39, antisense, 59-ACCTTTCAAATGCAGTA- GATTCA-39), mouse IL12p40 (sense, 59-AGATGAAGGAGACAGAGGAG- 39,antisense,59-GGAAAAAGCCAACCAAGCAG-39), and b-actin (sense, 59-CGCGTCCACCCGCGAG-39, antisense, 59-CCTGGTGCCTAGGGCG- 39). The results were normalized to b-actin. The amount of each transcript was expressed by the formula 2ΔCt, where ΔCt = Ct of b-actin 2 Ct of gene, with Ct being the point at which fluorescence rises appreciably above background fluorescence.

Statistical analysis Prism software (GraphPad Software, San Diego, CA) was employed for statistical analysis. A Mann–Whitney U test was used to compare two experimental groups. To compare three or more experimental groups, a Kruskal–Wallis test followed by Dunn’s multiple comparison test was used for nonparametric data, and a one-way ANOVA test followed by Bonferroni’s multiple comparison test were used for parametric data. A p value ,0.05 was considered to be statistically signiﬁcant.

Results BV triggers innate and adaptive immune responses through TLR9/MyD88- and STING-dependent pathways BV is an enveloped dsDNA virus that is pathogenic for insects but cannot replicate in mammals, although it can transduce them (35). FIGURE 1. Role of TLR9, MyD88, and STING in the systemic in- 2/2 We have previously established the strong adjuvant properties of flammatory cytokine triggered by BV. WT (C57BL/6), TLR9 , and MyD882/2 mice (A) or STING+/+ and STING2/2 littermate mice (B) re- BV, which promotes the in vivo maturation of DCs and the produc- ceived a single injection of BV and their sera were titrated for the indicated tion of inflammatory mediators through mechanisms primarily medi- cytokines by ELISA. The results are expressed as the means 6 SEM for ated by IFNs-I, leading to potent humoral and CTL responses six (A) or five (B) mice per group. Data are representative (A) or are cu- against a coadministered Ag (29). Treatment of BV with BEI, which mulative from two independent experiments (B). (A)*p , 0.05, **p , alkylates selectively oligonucleotides (ODNs) but not proteins, 0.01, ***p , 0.001 (WT versus TLR92/2 and WT versus MyD882/2); (B) strongly decreases the ability of BV to induce IFN-I and to prime *p , 0.05, **p , 0.01, ***p , 0.001, ****p . 0.0001. 1154 CELLULAR SOURCES OF IFNs-I FOR CTL PRIMING

DNA viruses (36). To assess the role of STING in the systemic production of inﬂammatory cytokines triggered by BV, we injected this virus to STING+/+ and STING2/2 littermate mice. Strikingly, whereas the IL-12p40 production was fully independent of STING, the production of IFN-a/b,MIG,andIFN-g was strictly dependent on this pathway (Fig. 1B). The fact that both TLR9/MyD88- and STING-mediated pathways were involved in the BV-induced IFN-g and MIG responses (Fig. 1) suggests that the production of these inﬂammatory mediators can be positively cross-regulated by TLR9/ MyD88- and STING-derived signals. Although there exist some differences between TLR92/2,MyD882/2,orSTING2/2 mice and WT mice in the percentage of certain DC and macrophage subsets (Supplemental Fig. 2A–C), these differences cannot explain the different cytokine responses induced by BV in these mouse strains. Therefore, both TLR9/MyD88- and STING-dependent pathways are involved in the BV-induced innate immune response, being the systemic production of IFNs-I to this virus totally dependent on STING.

Inﬂammatory responses produced by cDCs and pDCs after Downloaded from in vitro stimulation with BV To compare the ability of cDCs and pDCs to secrete inﬂammatory cytokines after stimulation with BV, we cultured equal numbers of cDCs and pDCs isolated from the spleens of 129Sv mice with BV. 129Sv mice were chosen owing to their high pDC frequency in the

spleen (22). Upon stimulation with BV, pDCs secreted much http://www.jimmunol.org/ higher amounts of IFN-a/b and IL-12p70 than did cDCs, whereas cDCs were higher producers of IL-12p40 than were pDCs FIGURE 2. In vitro inflammatory cytokines produced by pDCs and (Fig. 2A). The induction of this cytokine profile was due to the cDCs after stimulation with BV. (A) FACS-sorted splenic cDCs and pDCs virus and not to any possible Sf9-derived contaminants because from 129Sv mice were stimulated with BV, and the cytokine production only the supernatant from BV-infected Sf9 cells, but not that from was determined in culture supernatant by ELISA. (B) pDCs and cDCs were uninfected cells, was able to stimulate the production of IFN-a stimulated with supernatants (SN) (diluted 1:10) from Sf9 cells infected or and IL-12p40 (Fig. 2B). Treatment with UV light, BEI, or Triton not with BV, or with BV (107 PFU/ml) either untreated or treated with UV X-100 impaired the capacity of BV to stimulate the production of light (BV-ultraviolet), BEI (BV-BEI), Triton X-100 (BV-Triton), or Ben- IFNs-I and IL-12p40 by pDCs and cDCs, whereas Benzonase zonase (BV-Benzo). As a control, cells were also cultured with a volume of by guest on September 29, 2021 treatment did not (Fig. 2B), suggesting that both the infectivity of Benzonase (Benzo) equivalent to the volume of Benzonase-treated BV. the viral particle and its encapsulated nucleic acids are critical for Data are representative of at least two independent experiments. the BV-induced stimulation of pDCs and cDCs. Indeed, BV-induced production of IFN-a/b by splenic cDCs was Role of TLR9/MyD88 and STING in the BV-triggered abrogated by treatment with UV light, Triton X-100, and BEI stimulation of cDCs and pDCs (Supplemental Fig. 3B), suggesting that the way in which the We then analyzed the PAMP receptors used by cDCs and pDCs to encapsulated nucleic acids are internalized is crucial for the BV- 2/2 sense BV. BM-derived or splenic cDCs and pDCs from TLR9 , triggered STING-dependent production of IFNs-I by cDCs. MyD882/2, TRIF2/2, and STING2/2 mice,aswellasfromtheir respective WT mice, were stimulated with BV and analyzed for Role of the TLR9/MyD88 pathway in the in vivo BV-induced cytokine production. Independently of whether they were splenic or cytokine production BM-derived, the highest levels of IFN-a/b and IL-12p40 were To assess the respective roles of cDCs and pDCs, as well as of the produced by pDCs and cDCs, respectively (Fig. 3, Supplemental TLR9/MyD88- and STING-mediated pathways, in the in vivo BV- Figs. 1B, 3A). For both DC subsets, the BV-induced production of triggered cytokine response, we analyzed the expression of IFN-a/b IFN-a and IL-12p40 was independent of Trif (Supplemental Fig. and IL-12p40 in splenic cDCs and pDCs isolated from BV-injected 1B). Interestingly, whereas the BV-triggered IFN-a/b production mice. As depicted in Supplemental Fig. 3C, expression of IFN-a was highly dependent on TLR9/MyD88 signaling in pDCs (Fig. and IL-12p40 by intracellular staining was only detected in pDCs or 3B, Supplemental Fig. 3A), it was strictly dependent on STING in cDCs, respectively. In every case, this expression was dependent on cDCs (Fig. 3C). In contrast, the BV-induced IL-12p40 production TLR9 (Supplemental Fig. 3C) and MyD88 (data not shown). Next, was dependent on TLR9/MyD88 signaling in both cDCs and pDCs we assessed the expression of IFN-a/b and IL-12p40 by quantita- (Fig. 3A, Supplemental Fig. 3A). Therefore, the involvement of tive RT-PCR in splenic cDCs and pDCs from WT, MyD882/2,and TLR9/MyD88 and STING in the BV-triggered innate response is STING2/2 mice that were previously injected with BV (Fig. 4A). cytokine and DC type-specific. Accordingly to the results obtained in vitro, we found that pDCs and Although less efficiently than intact virus, purified BV-DNA also cDCs were, respectively, the highest producers of IFN-a/b and of triggered the production of IL-12p40 in both cDCs and pDCs, as IL-12p40 in response to BV infection (Fig. 4). The expression of well as the production of IFN-a/b in pDCs, and this response was IFN-a/b in pDCs and the production of IL-12p40 in cDCs were TLR9/MyD88-dependent (Supplemental Fig. 3A). However, BV- dependent on MyD88 (Fig. 4B) and independent of STING (Fig. DNA hardly stimulated the production of IFN-a/b in cDCs. This 4C). Low but detectable levels of IFN-a and IFN-b in response to finding could be due to a less efficient internalization of the free BV were detected in WT cDCs by quantitative RT-PCR, which viral DNA in comparison with that of the virus-packaged DNA. were similar to those of MyD882/2 cDCs (Fig. 4B). We could not The Journal of Immunology 1155

FIGURE 3. Role of TLR9 and STING in the in vitro production of inflammatory cytokines by pDCs and cDCs in response to BV. (A) Experiment schedule. FACS-sorted splenic pDCs and cDCs from WT and TLR92/2 mice (B) or from STING+/+ and STING2/2 mice (C) were stimulated with BV (107 PFU/ ml). As control we also stimulated cells with CpGA-2216 (25 mg/ml) or R848 (1 mg/ml) (B and C). Cytokine production was determined in culture supernatants by ELISA. Data are representative of two indepen- Downloaded from dent experiments. http://www.jimmunol.org/ detect IFN-a expression by cDCs in either STING+/+ or in STING2/2 In vivo cDCs are responsible for the systemic inflammatory littermate mice (Fig. 4C). However, a BV-triggered IFN-b response response triggered by BV +/+ was clearly detectable in STING mice, which was totally abro- The predominance of cDCs in different tissues (22) (Supplemental 2/2 gated in STING mice (Fig. 4C). Therefore, the involvement of Fig. 2D) may strongly affect the importance of these cells in the the TLR9/MyD88- and STING-mediated pathways in the in vivo systemic inflammatory response induced by BV. To evaluate the production of cytokines by BV is also DC- and cytokine type– participation of pDCs in the systemic response, we depleted these specific. More importantly, on a per-cell basis, pDCs are the main cells using an anti–mPDCA-1 mAb (12) before BV administration. by guest on September 29, 2021 in vivo producers of IFN-a/b, whereas cDCs are the main in vivo The efficient pDC depletion was confirmed by flow cytometry IL-12p40 triggers. (Fig. 5A). As shown in Fig. 5B, pDC depletion did not affect the

FIGURE 4. Role of MyD88 and STING in the in vivo production of inﬂammatory cytokines by pDCs and cDCs in response to BV. (A) Experi- ment schedule. Mice were left untreated or were injected (i.v.) with BV (107 PFU) and 90 min later spleens were harvested. Splenic cDCs and pDCs were FACS-sorted from a DC-enriched splenocyte preparation and processed for RNA isolation. (B and C). Expression of IFN-a, IFN-b, and IL- 12p40 mRNA in FACS-sorted splenic pDCs and cDCs from WT and MyD882/2 mice (B) or from STING+/+ and STING2/2 littermate mice (C) analyzed by quantitative RT-PCR. Data are representative of at least two independent experiments. 1156 CELLULAR SOURCES OF IFNs-I FOR CTL PRIMING systemic IFN-I response induced by BV, nor the level of expres- DTR mice. Administration of DT to these mice 24 h before BV sion of IL-12p40, IFN-g, or MIG. injection results in depletion of CD11c+ cells in the spleen (Fig. 5C, To examine whether cDCs played a role in the systemic inﬂam- 5D). The most affected populations were CD11chigh cDCs (depletion matory response triggered by BV, we used transgenic mice carrying .95%), whereas other CD11c+ cells were depleted to a lesser extent the DTR linked to the CD11c promoter (CD11c-DTR mice) (28). (macrophages, between 60 and 80%; NK cells, 50%; pDCs, 40%) Because nonhematopoietic cells may also express the CD11c-DTR (Fig. 5C, 5D). CD11cint cDCs and CD11c2 cells (such as T and transgene (28), we produced CD11c-DTR → WT chimeric mice in B lymphocytes and NKT cells) were not affected by this treatment. which only the hematopoietic compartment is derived from CD11c- AdministrationofDTtoCD11c-DTR→ WT chimeric mice 24 h Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. cDCs, but not pDCs, are required for the in vivo inflammatory response promoted by BV. (A) Percentage of pDCs (CD192NK1.12CD11c+ B220+Ly6C+) in total splenocytes 24 h after PBS, anti–mPDCA-1 mAbs, or rat IgG2b delivery. (B) Seric levels of cytokines triggered by BV in C57BL/6J mice that were previously treated with PBS, anti–mPDCA-1 mAbs, or rat IgG2b. (C and D) Absolute number of different subsets of DCs, macrophages (C), and lymphocytes (D) in total splenocytes from CD11c-DTR → WT chimeric mice 24 and 96 h after PBS or DT administration. Cells were gated as indicated in Supplemental Fig. 2A, and phenotypic features are shown in Supplemental Fig. 2B. M, macrophages. Data were compiled from two independent experiments (n = 6). *p , 0.05, **p , 0.01, ***p , 0.001. (E) Seric levels of cytokines triggered by BV in CD11c-DTR → WT BM chimeric mice that were previously treated with PBS or DT (24 or 96 h before virus injection). *p , 0.05, **p , 0.01, ***p , 0.001 (NT versus DT, 24 h); $$p , 0.01, $$$p , 0.001 (NT versus DT, 96 h); ##p , 0.01, ###p , 0.001 (DT [24 h] versus DT [96 h]). (B and E) Results are expressed as the means 6 SEM and represent cumulative data from four (B) and six (E) mice tested in two independent experiments. The Journal of Immunology 1157 before BV injection strongly reduced the serum levels of IFN-a, induced production of IFNs-I (40). We have confirmed these data IFN-b, IL-12p40, IFN-g, and MIG (Fig. 5E). Similar results using mice deficient for IPS-1, the adaptor molecule triggering ret- were obtained when we used CD11c-DTR mice instead of inoic acid–inducible gene-I– and MDA-5–mediated IFN-I induction CD11c-DTR → WT chimeric mice (data not shown). (41) and by depleting pDCs with anti–PDCA-1 mAb (Fig. 6A, 6B). Interestingly, the total number of CD11chigh cDCs returned to Note that the systemic production of IL-12p40 in response to normal levels after 96 h of DT treatment, whereas pDCs and LCMVArm was independent of both IPS-1 and pDCs (Fig. 6A, 6B). macrophages remained depleted (20 and 65–70%, respectively) To evaluate the contribution of cDCs to the IFN-I response (Fig. 5C, 5D). This property is commonly used to discern the role triggered by LCMVArm, CD11c-DTR → WT chimeric mice were of cDCs and/or macrophages in different biological process us- injected with DT 24 and 72 h before virus injection (48 and 96 h ing CD11c-DTR mice (37, 38). As depicted in Fig. 5E, CD11c- before the peak of the IFN-a response, respectively). As shown in DTR → WT chimeric mice treated with DT 96 h before BV Fig. 6C, the administration of DT 24 h before virus injection injection produced systemic levels of IFN-a,IFN-b,andIFN-g strongly reduced the seric levels of IFN-a and IL-12p40, whereas comparable to those in nontreated mice, although IFN-b and mice treated with DT 72 h before LCMVArm infection produced IFN-g values were slightly higher in the former group. Addi- normal levels of these cytokines. These data indicate that, as tionally, MIG levels reached significantly higher values in the demonstrated for BV, cDCs play an essential role in the in vivo group treated with DT 96 h compared with nontreated mice. This production of IFN-a, as well as of IL-12, in response to LCMV. effect could be due to a higher capacity of the newly differen- TLR-independent pathways control the systemic tiated cDCs to produce inflammatory mediators or to a release of virus–triggered IFNs-I response required for CTL activation the break that controls cDC-mediated cytokine response. Inter- Downloaded from estingly, the IL-12p40 response was only partially restored in Using IFNAR-deficient mice, we have previously shown that BV mice treated with DT 96 h before BV infection, suggesting that triggers a strong CTL response through mechanisms mediated other cells, likely macrophages, are involved in the BV-triggered primarily by IFN-a/b (29). In good agreement with these previous IL-12p40 response. Finally, because the number of NK cells was results, C57BL/6 mice treated with anti-IFNAR neutralizing restored 96 h after DT treatment, we cannot rule out the possi- mAbs and immunized with BOVAp in combination with BV eli-

bility that NK cells might also contribute to the BV-triggered cited a much weaker CTL response than did those treated with http://www.jimmunol.org/ IFN-g production. a control Ab (Fig. 7A). To assess the contribution of pDCs to the Taken together, these data indicate that despite their lower potency CTL response triggered by BV, pDC-depleted mice were immu- on a per-cell basis, cDCs play an essential role in the in vivo in- nized with BOVAp in combination with BV. The percentage of flammatory response triggered by BV, including IFN-a/b production. OVA-specific CTLs was not affected by pDC depletion (Fig. 7B). In contrast, the CTL response induced by BV in CD11cDTR → In vivo cDCs are responsible for the systemic inflammatory BL6 chimeric mice was almost fully abolished by DT treatment response triggered by LCMV 24 h before virus injection (Fig. 7C). This response, however, was To assess whether in vivo cDCs, in response to a virus that infects and rapidly restored, reaching levels even higher than those observed replicates in mice, also produce IFNs-I more efficiently than do pDCs, in nontreated mice when DT was given 96 h before BV. by guest on September 29, 2021 we selected the LCMVArm virus, which elicits an acute infection cDCs are both the main APCs for naive CD8 T cells and the main in mice (39). Although pDCs have been shown to detect LCMV source of IFN-I after immunization in the presence of BV. Thus, to through TLR7-dependent mechanisms (40), they have an early and clarify their role in the induction of CTL responses, we took ad- limited impact on the amplitude and duration of IFN-a responses vantage of the fact that the BV-triggered IFN-I production by cDCs to LCMV, contributing only to the IFN-I response detected is strictly dependent on STING, in contrast to the production by 18–24 h postinfection (40). Rather, still unidentified cells, activated pDCs, which is mediated by TLR9/MyD88. As shown in Fig. 7D, by a melanoma differentiation-associated protein 5 (MDA-5)– a smaller but still significant CTL response was observed in dependent pathway, seem to be the key source of the LCMV- TLR92/2 and MyD882/2 mice, in comparison with WT mice,

FIGURE 6. Role of IPS-1, pDCs, and cDCs in the systemic IFN-I response elicited by LCMV. (A) C57BL/6 (WT) and IPS-12/2 mice were infected (i.v.) with LCMVArm. (B) C57BL/6 mice received two i.v. injections (24-h interval) of anti–mPDCA-1 mAbs (or control rat IgG2b). (C) CD11c-DTR → WT BM chimeric mice received PBS or DT (i.p.). (B and C) Twenty-four hours after the ﬁrst mAb injection (B)or 24 or 72 h after the DT treatment (C) mice were infected (i.v.) with LCMVArm. (A–C) At the indicated times, the levels of IFN-a and IL-12p40 were determined in the sera by ELISA. The results are expressed as the means 6 SEM and represent the cumulative data from six mice tested in two independent experiments. (A and B)*p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. (C)*p , 0.05, **p , 0.01, ****p , 0.0001 (NT versus DT, 24h); #p , 0.05 (NT verus DT, 72 h); xp , 0.05, xxp , 0.01, xxxp , 0.001 (DT [24 h] versus DT [72 h]). 1158 CELLULAR SOURCES OF IFNs-I FOR CTL PRIMING

FIGURE 7. TLR-independent virus-triggered IFN-I production is the key IFN-I source for CTL priming. (A and B) C57BL/6 mice were left untreated or received two i.v. injections (24-h interval) of anti-IFNAR mAbs (or control mouse IgG1) (A) or anti–mPDCA-1 mAbs (or control rat IgG2b) (B). (C) CD11c-DTR → WT chimeric mice were left untreated or treated with a single injection of DT. Twenty-four and 96 h after the DT treatment mice were immunized with BOVAp alone or together with BV. (D–F) WT, TLR92/2, and MyD882/2 mice (D) or Trif+/+ and Trif2/2 (E)or STING+/+ and STING2/2 littermate mice (F) were immunized with BOVAp alone or together with BV. (A–F) The percentage of OVA-speciﬁc CD8 T cells/ total CD8 T cell was analyzed at day 7 in the spleen by b H-2K -OVA257–264-tetramer-PE staining. The results shown are cumulative data from four (A and B)orat least ﬁve (C–F) mice tested in two independent experiments. Data from control mice immunized with BOVAp or BV alone are also shown. ***p , 0.001, Downloaded from ****p , 0.0001. (G) WT and IPS-12/2 mice received four i.v. injections (24-h interval) of anti-IFNAR mAbs (or control mouse IgG1) or two i.v. injections (24-h interval) of anti–mPDCA-1 mAbs (or control rat IgG2b). (H) CD11c-DTR → WT chimeric mice were left untreated or treated with a single injection of DT. G H

( and ) Twenty-four hours after either the ﬁrst in- http://www.jimmunol.org/ jection of mAb (G) or DT treatment (H), and 72 h after the DT treatment (H), mice were infected (i.v.) with LCMVArm and 15 h later they were immunized (s.c.) with OVAp. The number per microliter of blood of OVA-speciﬁc CD8 T cells was determined at day 7 by b H-2K -OVA257–264-tetramer-PE staining. The results shown are cumulative data from at least four mice tested in two independent experiments. Data from control groups injected with OVAp or LCMVArm alone are also shown. (G) Because mice treated with by guest on September 29, 2021 control mouse IgG1 or rat IgG2a give comparable results, they were plotted together under the IgG alias. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

upon immunization with BOVAp plus BV. Trif was not required for LCMV clone 13 (40), pDC depletion by anti–PDCA-1 mAbs for the BV adjuvanticity (Fig. 7E). In contrast, in the absence of had no significant effect on the CTL response elicited by STING, the BV-triggered CTL response was markedly reduced LCMVArm (Fig. 7G). Interestingly, IPS-1 deficiency impaired the (Fig. 7F). Because STING2/2 mice have been shown to generate CTL response triggered by LCMVArm against OVAp (Fig. 7G), normal CTL responses following Listeria monocytogenes rein- confirming previous data obtained in MDA-52/2 mice (40). These fection (42), this lack of CTL response was not due to an intrinsic data, together with the findings that the LCMV-elicited IFN-I re- defect of these mice. Given the impaired BV-triggered IFN-I sponse was drastically reduced in the CD11c-DTR system (Fig. 6C) (Fig. 1B) and CTL (Fig. 7F) responses in STING2/2 mice, to- and in the absence of IPS-1 (Fig. 6A), point out cDCs as the most gether with the strict dependence of STING on the cDC-induced likely IFN-I cellular source for the LCMV-triggered CTL response. IFN-I production to BV (Figs. 3C, 4C) and the markedly reduced Importantly, the residual CTL response detected in IPS-12/2 mice IFN-I response prompted by BV in cDC-ablated mice (Fig. 5E), was strictly dependent on IFN-I-signaling and pDCs (Fig. 7G). our data strongly suggest that cDCs lead to the systemic IFNs-I These data stand for an underlying role of the pDC-induced IFN-I production required for the BV-induced CTL response. production in CTL activation, a function that becomes important We have also assessed the contribution of different IFN-I cellular only when the TLR-independent IFN-I response decline. sources and signaling pathways on the CTL response induced by As observed in the BV viral model, the OVA-specific CTL re- LCMVArm. To skip the effect of viral replication and Ag expression sponse induced by LCMV was also impaired by DT administration on CTL priming, we analyzed at day 7 of virus injection the CTL 24 h before virus injection (Fig. 7H). response induced against a nonrelated epitope, OVA257–264,administered as soluble peptide. The immunodominance of the LCMV Discussion epitopes was overcome by injecting a high dose of soluble Although it is well accepted that IFNs-I, either alone or in com- OVA257–264 peptide (OVAp) (500 mg/mouse). It is already known bination with other signal 3 cytokines, are critical for the generation that LCMVArm elicits a strong CTL response through mechanisms of CTL responses (3, 4, 43), the roles played by the diversity of highly dependent on IFN-I (6). We have confirmed this result using IFN-I cellular sources as well as by the various signaling pathways an anti-IFNAR blocking mAb (Fig. 7G). Additionally, as reported involved remain poorly understood. Identifying the cells and The Journal of Immunology 1159 pathways that shape the inflammatory milieu required for CTL virtually all CD11chigh cDCs are depleted from the spleen and priming would facilitate the development of improved vaccines reached comparable numbers to those in nondepleted mice after against intracellular pathogens and tumors. 96 h of DT treatment (the present study and Ref. 38). DT Through its capacity to activate both pDCs and cDCs by different causes cell death by apoptosis (47), which does not induce pathways, BV constitutes an interesting nonreplicative viral model inflammatory immune responses (48). Other CD11c+ cells are to evaluate the impact on CTL priming of various pathways and depleted by DT treatment, such as NK cells, pDCs, and mac- cellular sources of inflammatory cytokines, independently of viral rophages. However, the extent of their depletion is less than for replication and Ag expression. Indeed, BV is recognized by TLR9/ CD11chigh cDCs, and, in the case of pDCs and macrophages, MyD88- and STING-dependent pathways. Interestingly, whereas the the depletion persists beyond 96 h of DT injection. Our findings in vivo BV-triggered production of IL-12p40 is partially dependent that the systemic IFN-I production following BV and LCMV on TLR9/MyD88 (the present study and Ref. 44), the in vivo IFN-I injection was abolished in CD11c-DTR → BL6 chimeric mice production to BV is strictly STING-dependent. Moreover, the in- treated with DT 24 h before virus injection, but was restored to volvement of TLR9/MyD88- and STING-dependent pathways in normal levels when DT was given 72–96 h before virus, sensing BV is not only cytokine type–specific but also DC subset- strongly support the conclusion that CD11chigh cDCs, but not specific. In this regard we show that the BV-triggered production of other CD11c+ cells, are the major effectors of the IFN-I pro- IL-12p40 is dependent on TLR9/MyD88 in both pDCs and cDCs, duction in response to BV and LCMV. whereas the production of IFN-I is TLR9/MyD88-dependent in cDCs have also been identified as the main in vivo IFN-I–producing pDCs and STING-dependent in cDCs. The cytokine type–specific cells against adenovirus (46). Several factors may account for cDC involvement of the TLR9/MyD88 pathway depending on the DC superiority over pDCs in the systemic virus-triggered IFN-I response. Downloaded from subset may be explained by the fact that in cDCs, unlike in pDCs, Numeric superiority of cDCs over pDCs may be a crucial factor. In TLR9 triggering leads to the activation of NF-kB, which is neces- fact, total cDC numbers in the body by far exceed the number of sary to induce inflammatory cytokines such as IL-12, but not to the pDCs (the ratio of pDCs to cDCs in an average mouse spleen is activation of IFN regulatory factor-7, which is required for IFN-I ∼1:10) (the present study and Ref. 22). Additionally, other factors production (45). The precise mechanisms by which BV induces such as virus-induced selective recruitment of cDCs and/or higher

IFNs-I in a STING-dependent manner remain unknown. The acti- in vivo virus tropism for cDCs may also explain the difference. http://www.jimmunol.org/ vating ligands for STING are cytosolic cyclic dinucleotides pro- Importantly, by depleting pDCs in WT mice we show that pDCs duced by bacteria or by cellular cGMP synthase, a host cell are dispensable for the BV- and LCMV-triggered CTL priming, nucleotidyl transferase that directly binds dsDNA, and in response confirming previous data using toxin receptor-based pDC-ablated synthesizes cGMP. Our in vitro and in vivo data using BV treated mice and both acute (LCMVArm) and chronic (LCMV clone 13) with BEI or Benzonase strongly suggest that viral DNA is the strains (40). Rather, the virus-induced TLR-independent produc- decisive PAMP for the BV-induced STING-dependent production tion of IFNs-I by cDCs seems to be the key IFN-I source for the of IFNs-I (the present study and Ref. 29), and STING-triggering CTL response triggered by BV and LCMV, as evidenced by the should likely occur through cGMP synthesis. impaired virus-triggered IFN-I and CTL responses in STING2/2 Owing to their ability to produce high levels of IFN-I per cell, and IPS-12/2 mice, respectively, together with the markedly re- by guest on September 29, 2021 pDCs are usually considered to be the main IFN-I–producing cells duced IFN-I induction by these viruses in cDC-ablated mice. during viral infection. However, growing evidence suggests that Interestingly, in the absence of high systemic levels of IFNs-I, as the contribution of pDCs to the virus-triggered IFN-I response is happens in IPS-I2/2 mice infected with LCMV, a significant CTL quite limited and time-dependent, whereas the participation of response could still be detected. Wang et al. (40) have recently nonprofessional IFN-I producing cells through TLR-independent shown that the residual LCMV-triggered CTL response detected in pathways is higher than anticipated (13–15, 21, 40, 46). This the absence of MDA-5 signaling was not dependent on IL-12, the discrepancy is explained by the frequent use of in vitro or ex vivo other major signal 3 cytokine for CTLs. In the present study, we assays comparing the IFN-I–producing abilities of isolated pDCs show that pDC depletion and IFN-I signaling blockade abolish the and cDCs to infer the contribution of these cells to the in vivo remaining CTL response induced by LCMVArm in IPS-12/2 IFN-I response to viruses. Indeed, our in vitro and ex vivo studies mice. Our findings reveal an underlying role for the pDC-induced clearly show that BV triggers the production of IFN-I in both IFN-I response in effectively priming CTLs, which becomes rel- pDCs and cDCs and that pDCs are far more efficient than cDCs at evant only when the systemic and TLR-independent virus-trig- producing IFN-I on a per-cell basis. However, despite this well- gered IFN-I production is spoiled. established property, pDCs were highly dispensable for the in vivo Finally, the mechanism behind the detrimental effect of TLR9/ IFN-I response to BV, as was shown by depleting these cells with MyD88 deficiency on the BV-triggered CTL priming still remains anti–mPDCA-1 mAbs. pDC depletion has only an early and to be clarified. Given the fact that IFNAR blockade does not totally limited influence on the IFN-I responses to LCMV, confirming abrogate the CTL response prompted by BV (Ref. 29 and the previous data using toxin receptor–based pDC-ablated mice (40). present study), mechanisms independent of IFNs-I, such as IL-12 Additionally, using IPS-12/2 mice we have confirmed that the and/or BV-specific CD4 T cell help, could also be involved. Fi- MDA-5/IPS-1 signaling pathway has a major role in the IFN-I nally, because it has been described that TLR9 and MyD88 also response to LCMV. Therefore, other cells, through TLR- exert direct effects on T cell activation (49, 50), we cannot rule out independent pathways, are responsible for the in vivo IFNs-I the possibility that the diminished CTL response triggered by BV production in response to BV and LCMV. in TLR92/2 and MyD882/2 mice would be due to an intrinsic Unlike pDCs, which produce IFNs-I mainly upon TLR7/9 effect of these molecules on T cells. stimulation, IFNs-I are induced in most cells by TLR-inde- In conclusion, we show that in vivo, nonprofessional IFN-I– pendent pathways. Among them, cDCs have been reported to producing cDCs outpace pDCs and dominate systemic virus–trig- act as specialized IFN-I–producing cells in certain viral infec- gered IFN-I production by sensing the virus through TLR-in- tions (21, 46). CD11c-DTR transgenic mice are very useful to dependent pathways. Furthermore, the TLR-independent IFN-I assess the contribution of cDCs in the systemic virus–induced response is the key source of IFNs-I required for CTL activa- IFN-I response. In these mice, 18 h after a single DT injection, tion. Interestingly, when the virus-triggered TLR-independent IFN-I 1160 CELLULAR SOURCES OF IFNs-I FOR CTL PRIMING production is impaired, the pDC-induced IFN-I response becomes 19. Cervantes-Barragan, L., K. L. Lewis, S. Firner, V. Thiel, S. Hugues, W. Reith, relevant for CTL priming. Viruses often target the TLR-independent B. Ludewig, and B. Reizis. 2012. Plasmacytoid dendritic cells control T-cell response to chronic viral infection. Proc. Natl. Acad. Sci. USA 109: 3012–3017. pathways of IFN-I induction to replicate and survive in the host. 20. Honda, K., Y. Ohba, H. Yanai, H. Negishi, T. Mizutani, A. Takaoka, C. Taya, and The effect of pDC-induced TLR-dependent IFN-I response on CTL T. Taniguchi. 2005. 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