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IFN-α Induces APOBEC3G, F, and A in Immature Dendritic Cells and Limits HIV-1 Spread to CD4+ T Cells This information is current as of August 12, 2017. Venkatramanan Mohanram, Annette E. Sköld, Susanna M. Bächle, Sushil Kumar Pathak and Anna-Lena Spetz J Immunol 2013; 190:3346-3353; Prepublished online 20 February 2013; doi: 10.4049/jimmunol.1201184 http://www.jimmunol.org/content/190/7/3346 Subscription Permissions Email Alerts This article cites 62 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/190/7/3346.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017 References The Journal of Immunology IFN-a Induces APOBEC3G, F, and A in Immature Dendritic Cells and Limits HIV-1 Spread to CD4+ T Cells Venkatramanan Mohanram,1 Annette E. Sköld,1,2 Susanna M. Bächle, Sushil Kumar Pathak,3 and Anna-Lena Spetz I mmature myeloid dendritic cells (DCs) are present in the cervico-vaginal mucosa and have been proposed to be one of the first target cells during HIV-1 transmission (1). They are characterized by low expression of costimulatory molecules and high capacity for Ag uptake. Upon encounter with Ags capable of triggering myeloid DC maturation, DCs gain a migratory potential and decrease uptake of additional Ags. Subsequently, the myeloid DCs upregulate costimulatory signals required for effective T cell priming (2, 3). Immature myeloid DCs are susceptible to HIV-1 infection, which can lead to productive infection of the cells or hijacking by the virus (4). HIV-1 can use myeloid DCs as a Trojan horse, which leads to effective dissemination of the virus from the periphery to the local lymph nodes, enabling close contact with T cells (5). Although myeloid DCs can become infected with HIV1, they do not allow replication of the virus to a high extent, which may at least in part be explained by expression of restriction factors that can limit viral replication. Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, S-141 86, Stockholm, Sweden 1 V.M. and A.E.S. contributed equally to this work. 2 Current address: Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands. 3 Current address: Department of Genetics, Microbiology, and Toxicology, Stockholm University, Stockholm, Sweden. Received for publication April 23, 2012. Accepted for publication January 17, 2013. This work was supported by the European Commission (EC-FP7-242135 CHAARM and EC-FP6-037611 EUROPRISE), the Hedlunds Foundation, SLL-ALF, and the Swedish Foundation for Strategic Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Address correspondence and reprint requests to Dr. Anna-Lena Spetz, Center for Infectious Medicine, F59, Karolinska Institutet, Karolinska University Hospital Huddinge, S-141 86, Stockholm, Sweden. E-mail address: [email protected] Abbreviations used in this article: A3G, APOBEC3G; APOBEC3, apolipoprotein B mRNA–editing, enzyme-catalytic, polypeptide-like 3; DC, dendritic cell; env, envelope; MDDC, monocyte-derived DC; PEG, pegylated; Vif, Virion infectivity protein. Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201184 The apolipoprotein B mRNA–editing, enzyme-catalytic, polypeptide-like 3 family (APOBEC3) proteins comprise six members of cytidine deaminases (6). The expression of APOBEC3G (A3G) is elevated in monocyte-derived DCs (MDDCs) upon maturation, leading to suppression of reverse transcription and introduction of G-to-A hypermutations in the viral genome. Two other members of the A3 family, A3F and A3A, were also shown to be expressed in MDDCs and exert antiviral activity (6–8). Although humans have evolved powerful retroviral restriction factors, HIV-1 has acquired specific means to antagonize these antiviral defense mechanisms. The HIV-1 Virion infectivity protein (Vif) promotes virion maturation and infectivity and was shown to counteract the activity of A3G (6). In the absence of Vif, the antiviral effect of A3G is exerted via its incorporation into newly formed virions in the virus-producing cell. A successful incorporation leads to deamination of the subsequent viral DNA synthesized during the next round of the viral life cycle (9, 10). However, deaminase-independent mechanisms of viral inhibition were also described. In cases in which Vif is present in sufficient amounts, A3G is targeted to an E3-ubiquiting ligase complex, leading to its degradation in the virus-producing cell. Hence, there will be a race between the target cell and the incoming virus, in which the combat may depend on the expression levels of restriction enzymes already present upon encounter with the virus. There are examples of reports suggesting that A3 proteins may indeed exert an inhibitory effect directly on incoming viruses when the amount of Vif is relatively low (11, 12). In addition, as the HIV-1 replication in MDDCs is relatively low, this could theoretically give more time for the restriction enzymes to act. However, it is presently not fully explored how different members of the APOBEC family of proteins are regulated in MDDCs to achieve protection against HIV-1 infection. APOBEC family proteins can be induced by type I IFNs, such as IFN-a (6). There are several members of the IFN-a protein family, but they bind a common cellular receptor to exert antiviral activities (13). Different type I IFNs stimulate different anti- Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017 Cytokines and IFNs, such as TNF-a and IFN-a, upregulate costimulatory molecules in monocyte-derived dendritic cells (MDDCs), enabling effective Ag presentation to T cells. This activation of MDDCs is often accompanied by upregulation of apolipoprotein B mRNA–editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) (A3) family proteins that are able to restrict HIV-1 replication in MDDCs by inducing hypermutations in the viral genome. In this study, we show that TNF-a upregulates costimulatory molecules and are able to restrict HIV-1BaL replication in MDDCs without significant induction of A3G, A3A, or A3F. Conversely, low quantities of IFN-a failed to upregulate costimulatory molecules, did not induce IL-12p40 or migration, but significantly induced A3G, A3A, and A3F mRNA expression and restricted viral replication in MDDCs. We also showed that transmission of HIV-1 from MDDCs to autologous T cells was significantly reduced in the presence of IFN-a. Sequence analyses detected the induction of high frequency of G-to-A hypermutations in the env genes from HIV-1BaL–infected MDDCs treated with low quantities of IFN-a2b. These findings show that low quantities of IFN-a can induce functional A3 family proteins and restrict HIV-1 replication in MDDCs while keeping an immature nonmigratory phenotype, supporting further investigations of modalities that enhance retroviral restriction factors. In addition, the findings highlight the role of IFN-a as a double-edged sword in HIV-1 infection, and we show that IFN-a can be powerful in reducing HIV-1 infection both in MDDCs and T cells. The Journal of Immunology, 2013, 190: 3346–3353. The Journal of Immunology Materials and Methods Human subjects and blood collection Buffy coats from healthy human blood donors were obtained from the blood bank at Karolinska University Hospital Huddinge. Ethical approval was obtained from the medical ethics committee in Stockholm. MDDC cultures CD14+ monocytes were enriched from buffy coats by negative selection using RosetteSep Human Monocyte Enrichment (1 ml/10 ml blood; Stem Cell Technologies, Vancouver, BC, Canada). Monocytes were separated using Lymphoprep density gradient (Nycomed, Oslo, Norway) and were cultured for 6 d in MDDC medium containing RPMI 1640 (Life Technologies, Paisley, U.K.), supplemented with 1% HEPES [N-(2-hydroxyethyl)piperazine-N9-2-ethanesulfonic acid] (Life Technologies), 1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine (Life Technologies), 1% streptomycin (Life Technologies), 1% penicillin (Life Technologies), and 10% endotoxin-free FBS (Life Technologies) with the addition of recombinant human cytokines IL-4 (6.5 ng/ml; R&D Systems, Minneapolis, MN) and GM-CSF (250 ng/ml; PeproTech; London, U.K.) to obtain immature DCs (23). Immature MDDCs were left untreated (medium) or treated either with LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO) or with TNF-a at single-dose increasing concentration (0.3, 3, 30 ng/ml; R&D Systems) or with different subtypes of IFN-a (pegylated [PEG] IFNa2b, IFN-a2a, IFN-aA/D) at single-dose increasing concentration (102, 103, 104 U/ml; IntronA; Schering-Plough, R&D Systems). MDDCs were analyzed by flow cytometer for CD80 and CD86 expression 2 d after IFN-a or TNF-a treatment. Phenotypic characterization of MDDCs MDDCs were stained with the following anti-human mAbs: CD1a (clone NA1/34; DAKO, Glostrup, Denmark), CD14 (clone TÜK4; DAKO), CD3 (clone SK7; BD Biosciences), CD80 (clone L307.4; BD Biosciences), and CD86 (clone 2331/FUN-1; BD Biosciences). Cell surface expression was measured by a FACSCalibur flow cytometer (BD Biosciences). MDDCs were gated on CD1a+, CD32, and CD142 cells, and 5 3 104 cells/sample were acquired. HIV-1 growth and preparation The HIV-1 isolate HIV-1BaL (which uses CCR5 receptors) was obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (National Institutes of Health, Bethesda, MD). HIV-1 isolates were grown in PBMC cultures stimulated with PHA (2.5 mg/ml; Sigma-Aldrich) and IL-2 (75 IU/ml; Chiron, Emeryville, CA). Primary virus was isolated from HIV-infected patient blood by coculture with PHAactivated donor PBMCs. To concentrate the virus and to minimize the presence of bystander activation factors in the supernatant that could induce MDDC maturation, virus stocks were ultracentrifuged (138,000 3 g for 30 min at 4˚C) (Beckman L-80 Ultra-centrifuge, rotor 70.1; Beckman Coulter, Fullerton, CA), and the virus pellets were resuspended in RPMI 1640 with 10% FBS to obtain a virus concentrate. The titers of virus stocks were 1.7 3 106/ml 50% tissue culture infectious dose for HIV-1BaL. HIV-1 infection of MDDCs For infection of MDDCs, 3000–6000 50% tissue culture infectious dose/ million cells of HIV-1BaL were used (19). The percentage of infected MDDCs was determined by intracellular p24 staining after 7 d of infection, as previously described (23, 24). Cells were first stained for cell surface markers (CD1a and CD86) and then fixed in 2% formaldehyde (SigmaAldrich). Cells were washed in saponin solution (PBS with 2% FBS, 2% HEPES, and 0.1% saponin; Sigma-Aldrich), to allow permeabilization of the cell surface membrane, and then incubated for 1–2 h at 4˚C with a p24 mAb (clone KC57; Coulter, Hialeah, FL) or the corresponding isotype control Ab. p24 expression was assessed by a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). Gates were set on CD1a+ cells, and 5 3 104 cells were acquired per sample. Supernatants from MDDC cultures were harvested 6, 8, and 10 d after HIV-1 infection, and RT activity was quantified using the Lenti-RTactivity kit (Cavidi Tech, Uppsala, Sweden). HIV-1 MDDC–T cell transfer assay Monocytes and CD4+ T cells were prepared from the same donors using RosetteSep enrichment kits (Stem Cell Technologies). The T cells were frozen at 280˚C in 10% DMSO, 20% FCS, and 70% complete RPMI 1640 medium. On day 6, MDDCs were infected with HIV-1BaL and treated with PEG IFN-a2b at single-dose increasing concentration (102, 103, 104 U/ml), whereas the T cells were thawed and cultured in complete RPMI 1640 supplemented with IL-2 (75 IU/ml; Chiron) for 2 d. Two days postinfection, the virus was thoroughly washed away from the MDDC cultures and the autologous IL-2–treated T cells were added to the MDDCs in a 2:1 ratio. Cells were cultured for 4 d in the presence or absence of PEG IFNa2b at single-dose increasing concentration (102, 103, 104 U/ml). The cells were then stained with CD1a, CD3, CD86, and p24 mAbs, and 1 3 105 cells/sample were acquired in a FACSCalibur. Cytokine detection Supernatants from HIV-1BaL cultures were treated with 0.5% Triton X-100 to inactivate free virus. Secretion of human IL-12p40 was thereafter measured with an ELISA kit (Mabtech AB, Stockholm, Sweden). Real-time PCR analysis of A3A, A3F, and A3G RNA was extracted using the RNeasy Mini Kit (Qiagen), either from freshly isolated MDDCs or from MDDCs treated with LPS or with TNF-a or with different subtypes of IFN-a, as described above. RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Amplification of A3A, A3F, A3G, and 18S RNA was performed using the 7500 Real-Time PCR System (Applied Biosystems) and FAM dye-labeled TaqMan MGB probes and primers (Applied Biosystems) (25, 26). We did a homology BLAST for the primer probe, which is highly specific to the above-mentioned A3 molecules. Cycle threshold values for A3 were normalized to the value for 18S in control experiments. Data are presented as fold changes in mRNA copy number in the MDDCs treated with cytokines as compared with mRNA in MDDCs cultured in medium only. HIV-1 env sequencing and analysis Total cell DNA was isolated from cells treated with PEG IFN-a2b, 72 h postinfection with a QIAamp DNA mini kit (Qiagen). HIV-1 envelop (env) V1–V5 gene region was amplified by performing high-fidelity nested PCR using an EasyA kit (Stratagene). The primers used were as follows: round 1 (product size 1415 bp), forward 59-TTGCAATAGAAAAATTCTCCTC-39, and reverse 59-CCTGGTGGGTGCTACTCCTA-39); round 2 (product size 1098 pb), forward 59-CCATGTGTAAAGTTAACCCC-39, and reverse, 59ATGAGGGACAATTGAGAAGTGTCTAG-39; PCR condition as described in (27). Amplicons were purified by using a high pure PCR product purification kit (Roche), and cloned into the TA cloning vector provided in a TOPO TA cloning kit (Invitrogen) in accordance with the manufacturer’s instructions. Plasmids were isolated using Genejet plasmid mini prep kit (Fermentas), and the plasmids were sent to Eurofins MWG (Operon, Germany) for sequencing. Primers M13 forward (59-GTAAAACGACGGCCAG-39) and M13 reverse (59-CAGGAAACAGCTATGAC-39) were used for sequencing. G-to-A mutations were analyzed by using the program Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017 viral, antiproliferative, and clinical responses (14). The IFN receptor is coupled to a tyrosine kinase, which phosphorylates STAT proteins (15). IFN-a and LPS were shown to induce MDDC maturation and upregulate A3G in MDDCs (16–18). We previously showed that TNF-a plays a significant role in HIV-1BaL replication restriction in MDDCs that were cocultured with activated apoptotic T cells (19). The coculture with activated apoptotic T cells also led to induction of MDDC maturation as measured by upregulation of costimulatory molecule CD80 and CD86 expression (19, 20). Induction of MDDC maturation was previously shown to be accompanied by induction of A3G and A3F expression in MDDCs (7), and maturation suppresses HIV-1 infection through multifaceted mechanisms that involve decreased viral fusion (4), a block of reverse transcription (21), and a postintegration restriction that have been proposed to exist at the transcriptional level (22). In this study, we set out to investigate whether inducers of MDDC maturation such as TNF-a and IFN-a result in similar induction of expression of A3G, A3F, and A3A with subsequent inhibition of HIV-1 replication. We also measured whether this treatment is accompanied by increased MDDC maturation in terms of expression of costimulatory molecules, secretion of IL-12p40, and migratory capacity, as these are central functional properties of MDDCs. 3347 3348 APOBEC3G, F, AND A INDUCTION IN IMMATURE DENDRITIC CELLS Hypermut 2.0 that is freely available at http://www.hiv.lanl.gov/content/ sequence/HYPERMUT/hypermut.html (28). Migration assay Transwell chambers (8 mm; BD Biosciences) were inserted into wells of 24-well plates containing 750 ml complete RPMI 1640 supplemented with CCL21 (250 ng/ml; R&D Systems) and MDDCs were added in the upper well. The cell concentration in the lower chamber was assessed after 4 h by trypan blue exclusion of nonviable cells in an Auto T4 Cellometer (Nexcelom Bioscience, Lawrence, MA). Results Induction of CD80 and CD86 expression in MDDCs after treatment with IFN-a or TNF-a MDDC maturation can be triggered by proinflammatory cytokines such as TNF-a and IFN-a (29). To investigate whether exogenously added TNF-a and IFN-a can trigger MDDC maturation in the present culture system, immature MDDCs were treated with TNF-a Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017 FIGURE 1. CD80 and CD86 expression as well as IL-12p40 secretion in human MDDCs. Human in vitro differentiated monocytes cultured for 6 d in the presence of IL-4 and GM-CSF were used as the source of human immature MDDCs. Immature MDDCs were exposed to HIV-1BaL, treated with cytokines for 48 h, and then analyzed for expression of CD80 and CD86 molecules by flow cytometry. Gates were set on live large CD1a+CD32 cells. LPS, which is a potent DC activator, was used as a positive control, and MDDCs cultured in medium only were used as a negative control. Immature MDDCs were cultured in medium alone or in the presence of LPS or with TNF-a at single-dose increasing concentration (0.3, 3, 30 ng/ml) or with different subtypes of IFN-a (PEG IFN-a2b, IFN-a2a, IFN-aA/D) at single-dose increasing concentration (102, 103, 104 U/ml) in at least three independent experiments. (A) The average percentages of CD80+ MDDCs 6 SD for 11 donors. (B) The average percentages of CD80+ MDDCs 6 SD for 7 donors. (C) The average percentages of CD86+ MDDCs 6 SD for 11 donors. (D) The average percentages of CD86+ MDDCs 6 SD for 7 donors. (E) MDDCs were treated with increasing concentrations of TNF-a (0.3, 3, 30 ng/ ml), PEG IFN-a2b (102, 103, 104 U/ml), or with LPS as a positive control, in the presence of HIV-1BaL. The secretion of IL-12p40 was measured by ELISA in supernatants taken at 24 h of culture. The data are presented as pg/ml 6 SD for 6 donors. Significant differences compared with medium control were assessed by the paired one-way ANOVA with Bonferroni´s multiple comparison test, and significance is indicated by **p , 0.01 and ***p , 0.001. or PEG IFN-a2b at single-dose increasing concentrations (0.3, 3, 30 ng/ml and 102, 103, 104 U/ml, respectively). We used a PEG IFN, as the addition of PEG may delay clearance of the protein and is used in the clinic for treatment of hepatitis C in combination with ribavarin (30). The MDDCs were collected and stained for CD80 and CD86 for flow cytometry analysis 48 h post–TNF-a or PEG IFN-a2b treatment. LPS served as a positive control. We detected a significant increase in the expression of CD80 and CD86 in a dose-dependent manner in the cells that were treated with TNF-a (Fig. 1A, 1C). Similar data were obtained for PEG IFN-a2b displaying significant induction of CD80 and CD86 after stimulation with the highest dose of PEG IFN-a2b, but not when using the lower concentrations (102 or 103 U/ml) (Fig. 1A, 1C). Additional phenotypic analyses showed induction of CD83 and HLADR using the highest concentrations of IFN-a or TNF-a. The 7aminoactinomycin D staining did not reveal any toxic effects on the cells even at higher concentration of the cytokines (data not shown). To investigate whether the pegylation may have affected The Journal of Immunology the IFN-a2b from inducing the expression of costimulatory molecules, we further treated MDDCs with non-PEG IFN-a (IFN-a2a, IFN-aA/D) at single-dose increasing concentrations (102, 103, 104 U/ml). We confirmed that different IFN-a subtypes (with or without PEG), at high doses, can induce expression of costimulatory molecules (Fig. 1B, 1D). Mature MDDCs produce cytokines to influence the subsequent adaptive immune response. One cytokine important for Th1 induction is IL-12, and we therefore investigated whether IL-12p40 release into the supernatant correlated with the expression of maturation markers following HIV-1BaL infection and single-dose increasing concentrations of TNF-a or IFN-a (0.3, 3, 30 ng/ml and 102, 103, 104 U/ml, respectively). The release of IL-12p40 correlated with TNF-a treatment, but was not induced by PEG IFNa2b (Fig. 1E). Significant high quantity of IL-12p40 was only induced after stimulation with the positive control LPS. Restriction of HIV-1BaL replication in MDDCs upon treatment either with TNF-a or IFN-a (Fig. 2A). Thus, our data suggest that both TNF-a and IFN-a can significantly reduce viral replication in MDDCs. Reduced HIV-1 transfer from MDDCs to autologous T cells in the presence of PEG IFN-a2b, but not TNF-a We next investigated the viral transfer from HIV-1–infected MDDCs to cocultured autologous IL-2–activated T cells. MDDCs were infected with HIV-1BaL and treated with increasing concentrations of PEG IFN-a2b (102, 103, 104 U/ml) or the highest concentration of TNF-a for 2 d. Thereafter, the cultures were extensively washed, to prevent further infection from unbound virus. IL-2–activated autologous T cells were added to the MDDCs in a ratio of 2:1 and either left untreated (Fig. 3A) or continuously treated with PEG IFN-a2b (Fig. 3B) for 4 d. The p24 expression was then investigated in both T cells and MDDCs. MDDCs pretreated with the highest dose of PEG IFN-a2b (104 U/ml) displayed significantly reduced viral transfer to T cells even without addition of new PEG IFN-a2b (Fig. 3A). An even more impressive reduction of viral transfer was measured if the PEG IFN-a2b (102, 103, 104 U/ml) treatment was continued also with the addition of T cells. A significant reduction of the viral transfer was measured for both the intermediate and the higher concentration of PEG IFNa2b (103 and 104 U/ml) (Fig. 3B). Similar results were obtained for the MDDC infection in the coculture (data not shown). However, pretreating the MDDCs with the TNF-a concentration that protected the MDDCs from HIV-1BaL infection (30 ng/ml) (Fig. 2A) did not significantly alter the viral transfer to autologous T cells as compared with nontreated control (Fig. 3). Hence, TNF-a protects MDDCs from high HIV-1BaL infection by the induction of maturation, but this is not protecting interacting T cells from the infection. However, PEG IFN-a2b protects MDDCs from high HIV1BaL infection without inducing a mature phenotype, and also protects interacting T cells, even in the absence of exogenously added PEG IFN-a2b. Therefore, we continued to compare the induction of host-restriction factors in MDDCs treated with either PEG IFN-a2b or TNF-a at single-dose increasing concentrations (102, 103, 104 U/ml and 0.3, 3, 30 ng/ml, respectively). PEG IFN-a2b, but not TNF-a, upregulates mRNA expression of A3G, A3F, and A3A IFN-a is known to upregulate A3 molecules that possess cytidine deaminase activity in various cell types (32, 33). A3 molecules are FIGURE 2. Frequency of HIV-infected MDDCs. Immature MDDCs were exposed to HIV-1BaL, in the presence of LPS or with TNF-a at single-dose increasing concentration (0.3, 3, 30 ng/ml) or with different subtypes of IFN-a (PEG IFN-a2b, IFN-a2a, IFN-aA/D) at single-dose increasing concentration (102, 103, 104 U/ml). The percentage of infected MDDCs was assessed by flow cytometry analysis of intracellular p24 staining after 6–7 d in at least three independent experiments. (A) The average percentage of p24+ MDDCs 6 SD after 6–7 d after treatment with cytokines for 10 donors. (B) The average percentage of p24+ MDDCs 6 SD after 6–7 d after treatment with non-PEG IFN-a for 6 donors. Significant differences compared with HIV-1BaL– infected control were assessed by the paired one-way ANOVA with Bonferroni´s multiple comparison test, and significance is indicated by ***p , 0.001. (C) MDDCs were infected with HIV-1BaL. After 2 d, the cells were thoroughly washed and further cultured for 8 d. Supernatants from 4 donors were taken at 6, 8, and 10 d postinfection, and the reverse-transcriptase activity was measured with a Lenti RT activity kit. Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017 We previously showed that TNF-a plays a significant role in restriction of HIV-1BaL replication in MDDCs that are cocultured with activated apoptotic T cells (19). In addition, a-IFNs are known to play a major role in antiviral activity (13, 14, 31). To further investigate the role for TNF-a and IFN-a in inhibition of viral replication, HIV-1–infected MDDCs were treated either with TNF-a at single-dose increasing concentration (0.3, 3, 30 ng/ml) or with three different subtypes of IFN-a (PEG IFN-a2b, IFN-a2a, IFNaA/D) (102, 103, 104 U/ml), respectively. The cells were collected and quantified for expression of the HIV-1 structural protein p24 by flow cytometry 6 d after TNF-a and IFN-a treatments (Fig. 2A, 2B). The release of viral particles into the supernatant of infected cells did not increase after this time point (Fig. 2C). We have previously shown that the intracellular detection of p24 requires de novo synthesis of viral proteins and fails to detect capture of HIV-1 virions (23). There was large variability between donors with regard to HIV-1 infection efficiency, ranging from 8.9 to 59.4% after 6 d, as previously reported (19). However, there was a significant decrease in percentage of p24+ in cells treated with IFN-a in a dose-dependent manner (Fig. 2A, 2B). All three concentrations of different subtypes of IFN-a could effectively reduce p24 percentage. TNF-a, in contrast, could reduce the frequency of p24positive MDDCs only at the highest concentration (i.e., 30 ng/ml) 3349 3350 APOBEC3G, F, AND A INDUCTION IN IMMATURE DENDRITIC CELLS potent in inducing mutations of a broad spectrum of retroviruses, including HIV-1 (34–37). A3A was recently reported to be expressed in MDDCs and inhibited early phases of HIV-1 infection (8). In this study, we investigated whether A3G, A3F, and A3A were upregulated in MDDCs exposed to HIV-1 and treated with TNF-a and PEG IFN-a2b at single-dose increasing concentration (0.3, 3, 30 ng/ml and 102, 103, 104 U/ml), respectively. RT-PCR was performed 24 h post–TNF-a and IFN-a treatment. There was a significant upregulation of A3G, A3F, and A3A, when the cells were treated with PEG IFN-a2b, even at the lowest concentration. However, no significant upregulation of these molecules was detected even at the higher concentration of TNF-a (Fig. 4). Hence, we confirm a previous study showing expression of A3A in MDDCs and further show that a low concentration (102 U/ml) of FIGURE 4. A3G, A3F, and A3A mRNA expression after treatment with TNF-a and PEG IFN-a2b. Real-time PCR was used to determine fold changes in mRNA for A3G (A), A3F (B), and A3A (C) in MDDCs exposed to HIV-1BaL and treated with LPS, TNF-a at single-dose increasing concentration (0.3, 3, 30 ng/ml), or with PEG IFN-a2b at single-dose increasing concentration (102, 103, 104 U/ml). mRNA levels were compared among MDDCs cultured in medium for 24 h. Data represent the mean 6 SD from four different donors. Significant differences compared with medium control were assessed by the nonparametric Mann–Whitney U test and are indicated as *p , 0.05. PEG IFN-a2b is sufficient to induce up to a 100-fold (range fold increase 9.5–285) increase of A3A as well as significant induction of A3G and A3F. PEG IFN-a2b induces G-to-A hypermutation in MDDCs infected with HIV-1BaL To investigate whether there were signs of A3 family protein activity, we screened for G-to-A hypermutation in the HIV-1 env genes that were amplified from the DNA obtained from the infected MDDCs treated with PEG IFN-a2b (10 2 U/ml). The env region was chosen for analyses because several studies have confirmed that A3 exhibits a pronounced 59 to 39 editing gradient, resulting in a particular susceptibility to A3 cytidine deaminase activity (38, 39). Sixty different clones were sequenced, and the total numbers of bp investigated were 51,000. We observed a high frequency of G-to-A hypermutations in the DNA that were obtained from the cells treated with the relatively low concentration Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017 FIGURE 3. Viral transfer from HIV-1–exposed MDDCs to autologous IL-2–activated T cells in the presence of TNF-a or PEG IFN-a2b. MDDCs were treated with PEG IFN-a2b at single-dose increasing concentration (102, 103, 104 U/ml) or with TNF-a at 30 ng/ml for 2 d. The cells were then extensively washed and thereafter cocultured with autologous T cells that had been treated with IL-2 (75 IU/ml) for 2 d. The PEG IFN-a2b was either washed away (A) or replenished in the same concentration as during the HIV-1 infection (B). After additional 4 d, the cultures were assessed for p24 expression and phenotypical markers in at least three independent experiments. The average percentage of CD3+ p24+ T cells 6 SD for 10 donors is displayed. Significant differences compared with HIV-1BaL–infected control were assessed by the paired one-way ANOVA with Bonferroni´s multiple comparison test, and significance is indicated by *p , 0.05 and **p , 0.01. The Journal of Immunology 3351 Table I. G-to-A mutation frequencies in HIV-1 env sequences obtained from HIVBaL-infected MDDCs treated with PEG IFN-a2b Item Analyzed MDDC PEG IFN-a2b–Treated 102 U/ml (env V1, V2, V4, V5) Total no. of clonesa Total no. of bp Total no. of clones Fisher exact p value ,0.05 Total no. of G-to-A mutations No. of G-to-A mutations (GG context) No. of G-to-A mutations (GA context) Average no. of G-to-A mutations/100 bp MDDC PEG IFN-a2b–Treated 102 U/ml (env V3 Region) 60 33,240 9 60 17,760 5 1,615 496 831 4.85 860 245 360 4.84 MDDC PEG IFN-a2b–Treated 102 U/ml (Total env) 60 51,000 14 2,475 741 1,191 4.85 a Representative clones from three donors were sequenced. cells were used as a background control, and LPS-treated cells were used as a positive control for migration. For some donors, CCL21 was instead added in the top chamber as a control, but the number of migrating cells did not differ from untreated cells (data not shown). In accordance with the relative lack of costimulatory molecules, MDDCs treated with PEG IFN-a2b did not migrate toward the CCL21 gradient (Fig. 5). The LPS-treated cells, however, migrated efficiently after pretreatment, but not when stimulated just prior to the migration assay. MDDC migration is not induced by PEG IFN-a2b treatment In this study, we measured the induction of A3G, A3F, and A3A antiviral restriction factors and activation/maturation of MDDCs after HIV-1 infection and treatment with TNF-a or IFN-a. Our study shows significant inhibition of HIV-1BaL replication and induction of A3G, A3F, and A3A in MDDCs after incubation with relatively low concentration of PEG IFN-a2b (102 U/ml). The lower PEG IFN-a2b concentrations of 102 U/ml and 103 U/ml did not induce MDDC maturation, as we did not detect significant induction of the costimulatory molecules CD80 and CD86, or secretion of IL-12p40. The presence of 103 U/ml PEG IFN-a2b was, however, sufficient to protect autologous IL-2–activated T cells from high HIV-1 infection in a MDDC–T cell transmission setting. None of the investigated PEG IFN-a2b concentrations induced a migratory phenotype in MDDCs. However, higher concentrations of PEG IFN-a2b induced expression of costimulatory molecules and a dose-dependent induction of APOBEC3 family proteins. Notably, the relatively low concentration of 102 U/ml was sufficient to induce significant G-to-A mutations (4.85 per 100 bp) in env sequences obtained from the MDDCs. Cell-free HIV-1 virions can be relatively poor inducers of type I IFN production compared with HIV-1–infected cells, as suggested in a recent study showing that HIV-1–infected cells induced 10– 100 times more IFN-a (mean 600 U/ml) than cell-free HIV-1 virions in vitro (40). It remains to be elucidated whether a local IFN-a release at the site of transmission, without concomitant proinflammatory responses, can induce sufficient restriction factors to protect from infection. In a recent study, significant induction of A3G and A3F as well as reduction (20.921 [60.858] log10 copies/ ml) of viral load was demonstrated in HIV-1/hepatitis C–coinfected patients undergoing treatment with PEG IFN-a and ribavarin (30). The plasma concentrations measured in patients after s.c. injection of PEG IFN-a are in the same order (mean week 1, 1710 pg/ml or week 4, 2380 pg/ml) (41, 42) as the lower doses used in the current study (102 U/ml equals 1430 pg/ml PEG IFN-a2b). However, it remains to be elucidated whether PEG IFN-a can effectively penetrate into tissue compartments to modulate HIV-1 restriction factors in DCs and macrophage reservoirs. Although IFN-a has antiviral activity and can induce factors that limit viral replication (such as APOBEC molecules, TRIM-5 a, tetherin, and SAMHD1) Because PEG IFN-a2b did not induce strong maturation of MDDCs, but upregulated sufficient amounts of A3 molecules to reduce HIV-1 infection, we investigated whether a migratory phenotype was induced by the cytokine. MDDCs were treated with single-dose increasing concentrations (102, 103, 104 U/ml) of PEG IFN-a2b, either overnight or directly before addition to the upper well in a Transwell setting (Fig. 5). To stimulate migration of activated cells, the medium below the Transwell contained the chemokine CCL21, and the MDDCs were allowed to migrate for 4 h before the number of migrated cells was counted. Untreated FIGURE 5. Migration of MDDCs toward CCL21. MDDCs were treated with PEG IFN-a2b at single-dose increasing concentration (102, 103, 104 U/ml) either overnight (o.n.) or directly before addition to Transwells. LPS-treated cells were used as positive control and medium treated as a negative control. The lower wells contained CCL21 as a migratory gradient. The number of migrated cells in the lower chamber was counted 4 h after addition of cells. Data represent the mean 6 SD from eight different donors analyzed in at least three independent experiments. Significant differences compared with medium control were assessed by the nonparametric Wilcoxon matched-pairs signed rank test and are indicated as **p , 0.01. Discussion Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017 of PEG IFN-a2b (Table I). There was a similar distribution within the V3 env region as compared with other variable parts of env, in total, 14 of the 60 clones harbored significant mutation rates (Fisher exact p value ,0.05). The total numbers of G-to-A mutations were 2,475, and of these 741 were in GG context, whereas 1,191 were in GA context. The average number of G-to-A mutations per 100 bp was 4.85. Our data suggest that even a relatively low concentration of PEG IFN-a2b, which is not able to induce full MDDC maturation, is yet able to inhibit viral replication by upregulating A3 molecules, which in turn induce G-to-A hypermutation in the viral genome, causing the viruses to become replication incompetent. 3352 APOBEC3G, F, AND A INDUCTION IN IMMATURE DENDRITIC CELLS that patients with low viral set point had significantly higher levels of A3G mRNA both pre- and postinfection compared with patients that have high viral set point (56). Furthermore, HIV-1–exposed seronegative individuals were reported to have higher A3G expression levels than healthy controls (58), and PBMCs of HIV-1– exposed seronegative individuals have higher A3G expression and decreased susceptibility to infection of HIV-1 in vitro (59). Hence, it remains to be further explored which levels of APOBEC family proteins are required for effective block of HIV-1 acquisition in vivo and means to induce their expression without providing fuel for HIV-1 infection. It can be noted that highly exposed seronegative women were reported to have upregulated IFN-a expression in the cervix as quantified by immunohistochemistry (60). In addition, immunomodulatory or immunization strategies inducing A3G expression were reported to –correlate with reduced viral load or protection against infection in macaques (26, 61, 62). In this study, we showed that relatively modest quantity of IFN-a can significantly induce expression of A3G, A3F, and, in particular, A3A, without inducing signals that induce MDDC maturation and migration in vitro. We also showed that transmission of HIV-1 from MDDCs to autologous T cells is significantly reduced in the presence of IFN-a. This study supports future development of interventions that can enhance APOBEC expression and suggests that this can be achieved in myeloid DCs without inducing maturation and migration. Disclosures The authors have no financial conflicts of interest. References 1. Piguet, V., and R. M. Steinman. 2007. The interaction of HIV with dendritic cells: outcomes and pathways. Trends Immunol. 28: 503–510. 2. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245–252. 3. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Théry, and S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. Immunol. 20: 621–667. 4. Cavrois, M., J. Neidleman, J. F. Kreisberg, D. Fenard, C. Callebaut, and W. C. Greene. 2006. Human immunodeficiency virus fusion to dendritic cells declines as cells mature. J. Virol. 80: 1992–1999. 5. Coleman, C. M., and L. Wu. 2009. 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In natural hosts of SIV (such as African Green Monkeys and Sooty Mangabeys), the infection is often nonpathogenic despite the presence of high viral load (48). Experiments showed that type I IFN production was only transient after viral inoculation in natural hosts that do not develop disease (49–51). In contrast, in macaques in which SIV infection is pathogenic, there was a local accumulation of plasmacytoid DCs in the mucosal tissue shortly after primary infection, producing high quantities of IFN-a that can fuel the infection. It is possible that similar events take place during HIV-1 transmission and acute infection, which may be associated with high production of IFN-a and proinflammatory cytokines (47). High production of IFN-a and proinflammatory cytokines such as TNF-a will induce myeloid DC maturation and trigger migration of myeloid DCs into lymph nodes facilitating dissemination of the infection (1). The finding reported in this study suggests that a window of low IFN-a concentration can inhibit viral replication without inducing MDDC maturation and migration. Furthermore, the intermediate concentration of PEG IFN-a2b significantly prevented transmission of HIV-1 from infected MDDCs to interacting IL-2–activated autologous T cells. TNF-a, in contrast, could only limit replication in MDDCs in a relatively high concentration (30 ng/ml) that also induced MDDC maturation, and this treatment did not protect interacting T cells from HIV-1 infection. None of the concentrations of TNF-a (0.3–30 ng/ml) that we used in the current study showed induction of APOBEC3 family proteins. The antiviral effect exerted by TNF-a in MDDCs is in sharp contrast with its proviral activity in other cell types, such as monocyte-derived macrophages (52). Studies aiming to elucidate the mechanism of how TNF-a is acting to limit replication in MDDCs may shed light on these cellular differences. Earlier reports showed that A3A is highly expressed in monocytes (53–55), and a recent report demonstrated expression of A3A in MDDCs (8). Our study confirms expression of A3A in MDDCs (range fold increase 9.5–285) and shows a high mutation frequency after treatment with 102 U/ml PEG IFN-a2b. The induction of A3A, A3G, and A3F occurred in a dose-dependent manner, but the relative induction of A3A was higher compared with A3G and A3F in MDDCs. Of note is that A3A is the only member of the APOBEC3 family that is preferentially expressed in myeloid cells compared with peripheral blood lymphoid cells (8). A3G induces G-to-A mutations preferentially in a GG context, A3F prefer a GA context, whereas A3A can induce mutations in GG as well as in GA contexts (6). In the current study, we sequenced 60 clones and in total 51,000 bp, 2,475 G-to-A mutations were revealed. The G-to-A mutations occurred in both GG (n = 741) and GA (n = 1191) contexts, and the current study did not determine the relative contribution of each APOBEC family member. We did, however, detect significant inhibition of HIV-1 replication in the DCs, and it is conceivable that the total proportion of proviral sequences mutated by A3 proteins is underestimated, because extremely hypermutated sequences may fail to replicate and amplify by PCR. Although the treatment with low quantity of PEG IFN-a2b in MDDCs led to lethal hypermutations and inhibited viral replication, as shown in the present study, there is the theoretical possibility that APOBEC-mediated hypermutations may contribute to viral adaptation and even be involved in the acquisition of immune escape mutations early in HIV-1 infection (56). 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Pegylated 3353