TLR9 and TLR7 agonists mediate distinct type I IFN responses in humans and nonhuman primates in vitro and in vivo

TLR9 and TLR7 agonists mediate distincttype I IFN responses in humans and

nonhuman primates in vitro and in vivoMontserrat Puig,* Kevin W. Tosh,* Lynnsie M. Schramm,† Lucja T. Grajkowska,*

Kevin D. Kirschman,† Cecilia Tami,* Joel Beren,‡ Ronald L. Rabin,† and Daniela Verthelyi*,1

*Laboratory of Immunology, Division of Therapeutic Proteins, Center for Drug Evaluation and Research, †Laboratory ofBacterial, Parasite and Allergenic Products, and ‡Division of Veterinary Sciences, Center for Biologics Evaluation and Research,

Food and Drug Administration, Bethesda, Maryland, USA

RECEIVED JULY 22, 2011; REVISED SEPTEMBER 14, 2011; ACCEPTED OCTOBER 3, 2011. DOI: 10.1189/jlb.0711371

ABSTRACTHuman I-IFNs include IFN-� and 13 independently regu-lated subtypes of IFN-� (I-IFNs). TLR7 and -9 induce I-IFNs, but it is unknown whether their subtype repertoireis similar. This study used new PCR arrays that selec-tively amplify individual I-IFN subtype genes of humanand nonhuman primates to characterize the TLR7- and-9-mediated IFN response in vitro and in vivo. We showthat in human PBMCs, TLR7 agonists induce a rapidburst of I-IFN transcripts, consisting primarily of IFN-�1/13, -�2, and -�14. In contrast, TLR9 agonists, regardlessof the type used (CpG C-, B-, or D-ODN), promptedslower but sustained expression of IFN-�1/13, -�2, -�7,-�8, -�10, -�14, -�16, and -�21. These qualitative differ-ences were translated downstream as differences inthe pattern of IFN-inducible genes. In macaque PBMCs,imiquimod produced a short burst of IFN mRNA, domi-nated by IFN-�8, whereas C- or D-ODN induced agreater than tenfold increase in transcripts for all I-IFNsubtypes by 12 h of culture. Differences were more evi-dent in vivo, where TLR7 and -9 agonists induced sig-nificantly different levels of I-IFN transcripts in skin. Al-though the rates of gene transcription differed signifi-cantly for individual TLR9 agonists, their IFN-� subtypesignature was almost identical, indicating that the typeof receptor dictates the quality of the I-IFN response invitro and in vivo. These results may underlie the differ-

ential therapeutic effects of TLR7 and -9 agonists andshould inform future clinical studies. J. Leukoc. Biol. 91:147–158; 2012.

IntroductionI-IFNs play a key role in immunity, as they are expressed earlyin the innate immune response and directly, as well asthrough the induction of ISGs, amplify and direct the adaptiveimmune response [1]. I-IFNs can be produced by most cellsupon viral infection, but pDCs produce the highest amount inperipheral blood and skin [1, 2]. I-IFNs foster increased ex-pression of MHC and costimulatory molecules on monocytesand DCs, enable antigen cross-presentation, and facilitate viralclearing, making IFNs key in the protection against importanthuman pathogens such as HIV and HCV [3, 4]. In addition,the increased presentation of antigen, the up-regulation ofchemokines that attract immune cells to the site of injury, andthe activation of NK cells contribute to the IFN therapeuticeffect on cancer. Lastly, I-IFNs play an immunomodulatoryrole, promoting cell growth and survival and limiting inflam-mation [5].

In humans, I-IFNs include IFN-� and a group of 13 nonal-lelic IFN-� genes, which translate into 12 distinct IFN-� pro-teins [6, 7]. IFN-� subtypes share 75–99% of their amino acidsequence identity, bind to a common receptor (IFNAR1 andIFNAR2 heterodimer), and induce a largely overlapping co-hort of ISGs [8]. Although the different IFN-� subtypes appearto have risen through gene duplication and conversion [9],there is mounting evidence that the antiproliferative and anti-viral effect of the different subtypes varies and that there maybe a selective advantage to the host in maintaining 12 subtypesof IFN-� [8]. For example, studies have shown that IFN-�8and IFN-�10 are more effective at clearing viruses than IFN-�1[10–12].To date, however, a clear understanding of the ex-

1. Correspondence: Laboratory of Immunology, Division of Therapeutic Pro-teins, Center for Drug Evaluation and Research, Food and Drug Adminis-tration, Bldg. 29A, Room 3B19, 8800 Rockville Pike, Bethesda, MD 20892,USA. E-mail: [email protected]

Abbreviations: A–D-ODN�ODN types A–D, ACTB�actin-�,ATF5�activating transcription factor 5, AUC�area under the curve,Ct�comparative threshold, FDA�U.S. Food and Drug Administration,GBP1�guanylate binding protein 1, HBV/HCV�hepatitis B/C virus,HKG�housekeeping gene, I-IFN�type I IFN, IACUC�Institutional AnimalCare and Use Committee, IFNAR1/2�IFN-�R1/2, IRAK�IL-1R-associatedkinase, IRF�IFN regulatory factor, ISG�IFN-stimulated gene, LNA�lockednucleic acid, MET�c-Met proto-oncogene, Mx1�myxovirus (influenza vi-rus)-resistance 1, NMI�N-myc (and STAT) interactor, pDC-plasmacytoidDC, OAS1�2�-5�-oligoadenylate synthetase 1, qRT-PCR�quantitative realtime-PCR, SLC1A2�solute carrier family 1 glial high-affinity glutamate trans-porter member 2, TNFSF10�TNF superfamily 10

The online version of this paper, found at www.jleukbio.org, includessupplemental information.

Article

0741-5400/12/0091-147 © Society for Leukocyte Biology Volume 91, January 2012 Journal of Leukocyte Biology 147

pression and function of the IFN-� subtypes has been hin-dered by the absence of antibodies to accurately measure indi-vidual subtypes and the paucity of purified IFN-� subtypes tostudy their biological activity independently [6].

In recent years, numerous receptors that result in the induc-tion of I-IFNs have been identified, including TLR9 and -7,which are expressed in pDCs [1, 13]. TLR7 responds toguanosine/uridine-rich ssRNA, mediates the recognition ofseveral RNA viruses, including VSV, influenza, and HIV-1, andcan be selectively activated by small, synthetic antiviral com-pounds, such as imiquimod [14]. TLR9 is activated by ssDNAsequences that encode unmethylated CpG motifs, which aremore frequent in microbial DNA than in the mammalian ge-nome [15, 16]. Upon activation, the cytosolic portion ofTLR7 and -9 recruits the adaptor molecules MyD88, IRAK-1,IRAK-4, and TRAF6 to activate and translocate NF-�B orIRF7 to the nucleus to induce the transcription of inflam-matory cytokines, such as IL-6 and TNF-� or I-IFNs, respec-tively [17]. The precise rules that govern this dichotomy arenot well understood [18].

The development of synthetic molecules, such as imiquimodand CpG oligodeoxynucleotides (ODNs) which mimic TLRnatural ligands, has been essential to study the role of thesereceptors on the innate immune system. TLR9 is somewhatunique, in that although it is activated by well-defined, shortstrands of nucleic acids in a sequence-dependent manner, theODNs that bind and activate it have been classified into threedistinct groups based on their sequence, structure, and thebiologic response that they elicit [15, 16, 19, 20]: CpG ODNsthat have a phosphorothioate backbone, and several CpG mo-tifs have been termed B-ODN (CpG K-ODN in some publica-tions). These ODNs promote polyclonal B cell activation andmaturation of pDC but induce low levels of I-IFNs in PBMCs[15, 21, 22]. CpG D-ODN and A-ODN have a single CpG mo-tif, surrounded by a self-complementary core sequence andone (D-ODN) or two (A-ODN) poly-G strand ends that favorG-tetrad formation and ODN multimerization. D-ODNs stimu-late TLR9 on pDCs, but not B cells, to secrete high levels ofIFN-� [21]. Lastly, the CpG C-ODNs were designed to com-bine the activity and stability of the other two ODN types andcan mediate the induction of I-IFNs, as well as B cell activa-tion, but their potency in both effects is moderate [23]. It re-mains unknown how the different CpG ODNs lead to suchdistinct outcomes, although it has been postulated that thedifferential effects of B-, C-, and D-ODN depend on the sub-cellular organelle where the ODN activates TLR9. Thus, C-and B-ODN, which localize to late endosomes, preferentiallyinduce NF-�B activation, whereas D-ODNs, which are retainedin early endosomes, induce the production of I-IFNs [24–26].More recently, Sasai et al. [27] have suggested that adaptorprotein 3 mediates traffic of TLR9 and -7 to specialized lyso-somes, where they engage TRAF3 and activate IRFs to produceI-IFNs. The above studies suggested that the intracellular loca-tion of TLR, rather than the type of receptor, dictates the na-ture of its downstream signaling.

In trying to understand the selectivity of the immune re-sponse to TLR agonists, we have characterized the I-IFN re-sponse induced by synthetic agonists, which are recognized by

different receptors (TLR7 or TLR9) that share downstreamsignaling via MyD88 and have similar cellular distribution (pri-marily in pDC) or which activate the same receptor (TLR9) indifferent subcellular compartments (early and late endo-somes). Using novel arrays of primers to assess the known hu-man and rhesus macaque subtypes of I-IFNs, we show thatTLR7 and -9 agonists induce IFN responses that are qualita-tively and quantitatively different, whereas the different TLR9agonists induce a similar I-IFNs signature, despite significantdifferences in the magnitude of the IFN response.

MATERIALS AND METHODS

Isolation of human and rhesus macaque PBMCs andin vitro cell stimulationHuman PBMCs were provided by the Blood Bank of NIH (Bethesda, MD,USA) and separated by density gradient centrifugation over Ficoll-Hypaque,as described elsewhere [19]. Blood from Rhesus macaques was collected viafemoral venipuncture in Vacutainer acid citrate dextrose Solution A tubes(Becton Dickinson, Franklin Lakes, NJ, USA), as outlined in the IACUC-approved protocol. PBMCs were similarly separated by Ficoll-Hypaque gra-dients. Cells were cultured at a density of 2–4 � 106 cells/ml at 37°C inRPMI-1640 media, supplemented with 10% FCS and 100 U penicillin/mL,100 �g streptomycin/mL, glutamine, nonessential amino acids, Hepes, and�-ME, as described earlier [28]. CpG D35-ODN (D-ODN; 5�-ggTGCATC-GATGCAGGGGgg-3�), B2006 (B-ODN; 5�-tcgtcgttttgtcgttttgtcgtt-3�), andC2395 (C-ODN; 5�-tcgtcgttttcggcgcgcgccg-3�) were synthesized at the FDAcore facility (Rockville, MD, USA) and used at the concentration indicatedin each individual figure. Uppercase letters in the sequence indicate phos-phodiester backbone, as opposed to phosphorothioate linkages, indicatedby the lowercase characters. TLR7 agonist imiquimod and CL097 (Invivo-Gen, San Diego, CA, USA) were used at 10 �g/ml and 30 �g/dose, respec-tively. The endotoxin level of the TLR7 and -9 agonist solutions was deter-mined to be �0.1 EU/ml by the Limulus amoebocyte lysate test (Lonza,Walkersville, MD, USA).

In vivo administration of TLR agonists to rhesusmacaques and tissue collectionHealthy rhesus macaques (Macaca mulata; three to four animals/group)were obtained from Morgan Island, SC, USA. All studies were IACUC-ap-proved and conducted at the FDA Assessment and Accreditation of Labora-tory Animal Care vivarium on the NIH campus. Each animal received 3 i.d.doses of 300 �g CpG ODN (D-ODN or C-ODN) or 30 �g CL097 in thechest (two different sites, �10 cm apart) and the upper arm, using a 27-gauge � 5/8-inch hypodermic needle. Animals in the control group wereinjected with normal saline solution (Quality Biological, Gaithersburg, MD,USA). Skin biopsies (4 mm in diameter) were obtained at 0, 6, 24, and48 h postinoculation with biopsy punch (Miltex, York, PA, USA). Hemosta-sis was achieved using sterile gauze and digital pressure. The skin incisionwas closed with Vetbond (3M) surgical glue. The tissues were submerged inTrizol reagent (Invitrogen, Carlsbad, CA, USA) and frozen immediately.

Tissue homogenizationSkin biopsies were shredded by glass-bead friction. One millimeter-diame-ter Zirconia beads (BioSpec Products, Bartlesville, OK, USA) were added tothe samples in Trizol, prior to homogenization in the Mini-BeadBeater-8(Biospec Products). Each sample was homogenized for 30 s and thenrested on ice for 90 s, for five consecutive times. Subsequently, the sampleswere centrifuged for 15 min at 13,000 rpm. The Trizol reagent was trans-ferred to a new tube for RNA extraction.

148 Journal of Leukocyte Biology Volume 91, January 2012 www.jleukbio.org

RNA extraction, DNase treatment, and cDNAsynthesisTotal RNA was extracted from freshly isolated, cultured PBMCs (2–4 mil-lion cells) or skin biopsy homogenate using Trizol (Invitrogen), as permanufacturer recommendations. Upon resuspension of the total RNA, fur-ther purification of the mRNA from the skin samples was obtained withMicroPoly(A)Purist columns (Ambion, Austin, TX, USA). One microgramof total RNA or 0.5 �g mRNA was used to perform cDNA synthesis withthe high-capacity cDNA RT kit (Applied Biosystems, Foster City, CA, USA),according to the manufacturer’s instructions. For the analysis of IFN-� sub-type gene expression, 0.5–1 �g total RNA was treated with TurboDNase(Ambion) prior to cDNA synthesis, as per the manufacturer’s recommenda-tions.

qRT-PCRIndividual human gene expression assays. RNA (50–100 ng) was used

for qRT-PCR analysis with human or rhesus macaque-specific gene expres-sion assays and 2� Universal Master Mix (Applied Biosystems), as per themanufacturer’s instructions. For the analysis of human IFN-� and IFN-�subtype gene transcription, we amplified the mRNA with previously pub-lished [29] or newly designed primers (based on GenBank sequences andusing Primer Express 3.0 software) and Power SYBR Green PCR MasterMix (Applied Biosystems), as per the manufacturer’s instructions. Opti-mized primer sequences were selected based on amplicon size, meltingtemperature (Tm), primer-dimmer formation, and secondary structure.The specificity of the individual primer sets was confirmed by sequencingthe amplicon and by testing each set against individual IFN-� clones.Primer concentration was adjusted to eliminate primer-dimmer formationwithout decreasing the efficiency of the amplification of the template (seecomplete list in Supplemental Table 1). IFN-�4 transcripts were tested withthe corresponding human gene expression assay from Applied Biosystems.

Rhesus macaque I-IFN gene expression assays. Primer/probe se-quences that use molecular beacon [30], LNA [31], or standard linearprobes for measuring expression of rhesus IFN-� and IFN-� were designedusing Beacon Designer software (Premier Biosoft, Palo Alto, CA, USA).Only coding regions for mature proteins were entered for primer/probesearches. The performance of the primer/probe sets was enhanced by edit-ing the sequences or adding LNA oligonucleotide inhibitors. Each probewas conjugated to FAM and TAMRA or Black Hole quencher at the 5� and3� ends, respectively. Primer and probe sequences for the different IFNgenes are available upon request (unpublished results).

RT2 profiler PCR array system. Total RNA (500 ng) sample was addedto 550 �l 2� RT2 qPCR Mastermix (SABiosciences, Frederick, MD USA)and loaded into the IFN-�, -� response PCR array (SABioscience), as perthe manufacturer’s recommendations. This array contains a panel of 84ISG and five HKGs. The qRT-PCR human assays were conducted using a7900HT instrument (Applied Biosystems) with the following thermocyclingconditions: 94°C for 10 min, followed by 40 cycles of amplification at 94°Cfor 30 s and 60°C for 1 min. For the rhesus assay, the following conditionswere used: 50°C for 2 min, followed by 95°C for 10 min and 40 cycles of95°C for 15 s and 59.5°C for 1 min. GAPDH or 18S was used as HKGs tonormalize gene expression between samples. The most stable gene, used tonormalize the expression values of ISG genes, was identified using thegeNorm algorithm that considers Ct value variation in response to experi-mental conditions, as well as the expression level to calculate a stabilityscore. A score below 0.4 is considered to be very stable. Fold changes werecalculated by normalizing the expression using a HKG and then expressedas fold increase over unstimulated/untreated samples (2–��Ct method).

Human IFN-� subtype primers: specificity andsensitivitySpecificity of the individual subtype primer pairs was checked initially byamplicon sequencing. Briefly, upon in vitro stimulation of human PBMC(2 million cells) with 1 �M D-ODN for 12 h, total RNA was obtained, and1 �g was reverse-transcribed to cDNA using random primers and the first-

strand cDNA synthesis kit (GE Healthcare, Piscataway, NJ, USA), as per themanufacturer's instructions. To enrich the different IFN-� subtype ampli-cons, we amplified the cDNA with individual primer pairs specific for eachsubtype (see Supplemental Table 1), using the same PCR conditions as de-scribed in the previous paragraph. Amplicons were purified further withQIAquick PCR purification columns (Qiagen Sciences, Gaithersburg, MD,USA), and both strands of the final product were sequenced with nestedprimers with an homologous and specific sequence for each individualIFN-� subtype (sequences are available upon request). Sequencing dataconfirmed the purity of the individual amplicons (data not shown). Thespecificity of the assay was tested further using cloned IFN-� subtype 1, 5,7, 8, 10, and 21 DNA sequences (Geneservice, UK). Each clone was ampli-fied separately using a specific IFN-� primer set, following the protocol de-scribed above (data not shown). The assay sensitivity was evaluated basedon consecutive tenfold dilutions of the template, once the optimal concen-tration of each primer set was established (Supplemental Table 1). Dilu-tions were tested in triplicate, and the maximum sensitivity of the assay wasdetermined as the concentration of template at which consistently at leasttwo of the three triplicates had a Ct value �40 and no primer dimers.

ELISAIFN-�2 protein production was assessed by ELISA [32]. Briefly, Immunolon2 96-well microtiter plates (Dynex, Chantilly, VA, USA) were coated withanti-IFN-�2 antibody and blocked with PBS–5% BSA. Supernatants fromPBMC cultures were added and their IFN-�2 content quantified by addi-tion of the corresponding biotin-labeled anti-IFN-�2 detector antibody, fol-lowed by phosphatase-conjugated avidin and phosphatase-specific colori-metric substrate. Standard curves were generated using known amounts ofrecombinant human cytokine. All assays were performed in triplicate.

Statistical analysisRealTime Statminer® software v4.2 (Integromics®, Philadelphia, PA, USA)was used for the data and statistical analysis of the ISG expression in hu-man PBMCs. A heat map showing the hierarchical clustering was deter-mined based on the complete linkage method, whereas the similarity mea-sure was calculated using the Pearson correlation coefficient. The AUC foreach I-IFN subtype was calculated using the trapezoid rule as [yi(xi�1–x)�(1/2)(yi�1–yi)(xi�1–xi)], provided by Sigmaplot software (Systat Soft-ware, San Jose, CA, USA). The AUC for each subtype was added for eachsample to calculate the cumulative fold increase in gene expression in-duced over 48 h, and differences were tested using ANOVA.

RESULTS

Differential kinetics of IFN-�2 gene expression inPBMC stimulated with CpG ODN and imiquimodNucleic acids that stimulate pDCs are known to induce highlevels of endogenous IFN-�2. As shown in Fig. 1A, humanPBMCs stimulated with imiquimod (10 �g/ml) or D- orC-ODN (0.3 �M) induce similar maximal expression of a100,000-fold increase in IFN-�2 mRNA over basal levels. Thesame concentration of B-ODN (0.3 �M) yielded a weaker in-crease, in agreement with previously published studies [32,33]. Despite sharing signaling pathways and attaining similarlevels of transcripts, the kinetics of IFN-�2 induction by theagonists are markedly different, with imiquimod (or CL097,see Supplemental Fig. 1A) leading to a rapid increase inIFN-�2 mRNA, which peaked 2 h after stimulation and thendeclined sharply. In contrast, D-ODN showed a slow increasein IFN-�2 mRNA, which peaked after 12–18 h and remainedelevated for at least 24 h. An intermediate pattern was evidentfor C- and B-ODN, which peaked at 6 h and declined by 18 h.

Puig et al. IFN responses to TLR7 and -9 in vitro and in vivo

www.jleukbio.org Volume 91, January 2012 Journal of Leukocyte Biology 149

The differences in the kinetics of IFN-�2 mRNA expressionfor the different TLR agonists were not dependent on thedose of the TLR agonists, as cells stimulated with differentconcentrations of imiquimod or CpG ODN followed similarkinetics of expression (Fig. 1B). Therefore, ensuing studiesused 0.3 �M for TLR9 agonists and 10 �g/ml for imiquimod.The slower but sustained expression of IFN-�2 mRNA attainedwith D-ODN was associated with higher protein levels in cul-ture supernatants (Fig. 1). Cells stimulated with imiquimodproduced lower levels of protein, despite similar peak levels ofIFN-�2 transcripts, suggesting that the sustained expression ofthe IFN-�2 gene over time, rather than peak levels of RNA,correlates with higher protein expression (Fig. 1C and D). To-gether, these results show that TLR7 and -9 agonists inducedmRNA expression for IFN-�2, but the kinetics of expressionand the overall increase in the levels of mRNA and proteinvary depending on the stimuli.

TLR determines the IFN-� subtype expression profile

We next investigated whether TLR7 and -9 agonists inducedother I-IFN subtypes, as there are 13 nonallelic variants ofIFN-�, and each one is regulated by its own promoter. Specificprimers for each human IFN-� subtype were adapted from theliterature or developed in-house and validated for efficiencyand specificity (Supplemental Table 1, and data not shown).The gene expression of each subtype was subsequently as-sessed independently in blood samples of five healthy humandonors (Fig. 2A). Unstimulated PBMCs had undetectablemRNA levels of each one of the I-IFN genes, except for IFN-�,-�5, -�6, and -�17, which were expressed at low levels (Ct val-ues �32 and �36 for IFN-� and IFN-�, respectively; Fig. 2B).As shown in Fig. 2A, the kinetics of expression for I-IFNsbroadly mirrored those observed for IFN-�2 (Fig. 1A). Further,expression of IRF7 mRNA followed that of the I-IFNs, peaking

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Figure 1. Induction of IFN-�2 transcription and protein synthesis by TLR7 and -9 agonists. Human PBMCsfrom five healthy donors were stimulated in vitro with CpG D-, B-, or C-ODN (0.3 �M) or Imiquimod (10�g/ml) for 48 h. (A) Total RNA was isolated, reverse-transcribed, and subsequently analyzed by qRT-PCRusing IFN-�2-specific primers. Shown are geometric means of fold increases in mRNA expression over un-stimulated cells of the five donors. (B) Dose-dependent increase in IFN-�2 RNA levels in PBMCs stimulatedwith TLR7 and -9 agonists over time (data are representative of one of three experiments). (C) Cumulativefold increase in IFN-�2 over 48 h, measured as the AUC (see Materials and Methods). (D) IFN-�2 proteinlevels in the 48-h culture supernatants of human PBMCs (mean�sd).


at 12 h and 4 h for cells stimulated with TLR9 and -7 ago-nists, respectively (Supplemental Fig. 2). In contrast, the ex-pression of IRF9, which is known to play an important role inthe regulation of the IFN-feedback loop, peaked at the timewhen the expression of I-IFNs began to wane. No significantchanges over the baseline expression levels for IRF3 or IRF5were noted. Results show that the relative increase of each I-IFN subtype varied for TLR7 and -9 agonists. Cells activatedwith imiquimod predominantly up-regulated the expression ofIFN�1/13, -�2, and -�14 and did not up-regulate IFN-�4. Incontrast, although TLR9 agonist-treated cells showed preferen-tial up-regulation of IFN-�1/13, -�2, and -�14 at 2 h and 4 h,the transcription of all I-IFN genes continued to rise at 6 h(not shown) and 12 h (C- or B-ODN) or up to 24 h (D-ODN)before starting to decline. Of note, the relative increase in

IFN-�1, -�2, -�7, -�8,- �10, -�14, -�16, and -�21 expression ap-pears higher than the increase for IFN-�, -�5, -�6, and -�17, asa result of low but detectable expression levels in the unstimu-lated cells (Fig. 2B). However, for the four latter genes, theabsolute amplification values (Ct) at the peak of expressionwere comparable with the other subtypes, indicating that theTLR9 agonists induced or enhanced the up-regulation of allI-IFN subtypes. When assessed as the overall I-IFN response,calculated as the sum of the AUC of each individual gene dur-ing 48 h of culture, the increase in transcript expression in-duced by D-ODN was 3.6�, 130�, and 40� higher than inthe same cells stimulated with C-ODN, B-ODN, and imi-quimod, respectively (Fig. 2C). Interestingly, despite the differ-ence in total transcript increase, the relative contribution ofeach IFN-� species to the overall I-IFN response (shown as a

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Figure 2. IFN-� and IFN-� subtype expression in human PBMCs stimulated in vitro with imiquimod or CpG ODN. Transcript levels in PBMCsfrom five healthy donors stimulated with imiquimod or D-, C-, or B-ODN assessed at 0, 2, 4, 6, 12, 24, and 48 h using qRT- PCR. (A) Radar graphsare plotted in logarithmic (log10) scale using Excel (Microsoft, Redmond, WA, USA) and show fold increases of mRNA over the unstimulated cells(geometric mean of five independent experiments). (B) Baseline levels of IFN-�2 expression in unstimulated cells, normalized to the levels of 18S(HKG) of each sample (2–�Ct; where �Ct is the difference between the Ct of the gene of interest and the Ct of the HKG). (C) Cumulative foldincrease of I-IFN mRNA levels over 48 h was calculated as the sum of the AUC for each individual gene represented in A. (D) Relative contribu-tion of each subtype to the total increase in I-IFN mRNA transcripts over 48 h, expressed as percentage of the overall I-IFN response.



percentage) was very similar for the different TLR9 agonists,particularly B- and C-ODN (Fig. 2D): IFN-�1, -2, and -14 ac-counted for 50% of the total response, whereas IFN-�7, -�8,-�10, -�16, and -�21 accounted each for 10% of the tran-scription increases. In contrast, the IFN-� subtype responseelicited by imiquimod was markedly different, as IFN-�1, -�2,and -�14 subtypes accounted for �90% of the mRNA tran-scription, and the other subtypes accounted for �3% each(Fig. 2D and Supplemental Fig. 1B). Collectively, these data indi-cated that the type of the TLR agonist determined the magnitudeand kinetic of the response, but the receptor dictated the quality orsubtype signature of the I-IFN response induced.

Differential expression of ISG induced by TLRagonist treatmentsTo assess whether the qualitative differences in I-IFN subtype expres-sion had a biological effect, we next investigated whether it impactedon the expression pattern of ISG. For that purpose, we used a geneexpression array that included primers for 84 different IFN-relatedgenes. Not surprisingly, given that I-IFNs signal through a commonreceptor IFNAR, TLR7 and TLR9 activation significantly modulatedthe expression of an overlapping group of 30–35 genes out of the84 tested (greater than threefold change, and P�0.05; SupplementalFig. 3). Despite this, hierarchical gene clustering analysis with Pear-son correlation coefficient showed that the impact of the individualstimuli superseded that of intersubject variability (Fig. 3A). Thus,ISG expression in cells stimulated via TLR9 tended to segregate

from those activated with imiquimod. Moreover, among cells stimu-lated with TLR9 agonists, D-ODN segregated from those stimulatedby B- and C-ODN, suggesting that the individual TLR agonists im-pacted the relative expression of ISG mRNA.

Of the 30 genes that were significantly up-regulated by all ofthe TLR7 and -9 agonists, those encoding proteins with antivi-ral properties (OAS1, ISG20, bone marrow stromal cell anti-gen 2, and IFN-induced helicase C domain-containing protein1) seemed to be similarly up-regulated by imiquimod and CpGODN. In contrast, 11 genes with roles in cell mobilization(CXCL10, ACTB), apoptosis (TNFSF10), STAT-related signaltransduction (NMI), antigen presentation (HLA-DOA andHLA-DQA1), tissue regeneration (MET), and glutamate trans-porter (SLC1A2) showed significant differences in relative ex-pression between TLR9 agonists and imiquimod (Fig. 3B). Asthe array only analyzed 84 of the roughly 600 genes that aremodulated by IFNs, further studies will be needed to establishthe breadth of the difference between TLR7- and TLR9-in-duced ISGs. Nevertheless, these data suggest that the induc-tion of specific IFN subtype patterns via TLR7 and -9 may re-sult in subtle but important biological differences.

I-IFN expression pattern in PBMCs from macaquesMacaques and other nonhuman primates provide valuable sur-rogate models of human disease (including HBV, HIV, andcancer), where the outcome is linked to the IFN-� levels [34].Previous studies had shown that TLRs are highly conserved in

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Figure 3. Expression profile of ISG after TLR activation. mRNA expression of 84 ISGs in human PBMCs (n�5 healthy donors) stimulated withTLR7 and TLR9 agonists for 24 h was assessed by qRT-PCR using an IFN-�� response PCR array. Expression of 30 of 84 genes was increased sig-nificantly (greater than threefold; P�0.05) following stimulation with the TLR agonists (Supplemental Fig. 3). (A) Hierarchical clustering heatmap based on complete linkage method and Pearson correlation coefficient. (B) RNA fold increase ratio between levels of mRNA expression incells stimulated with CpG ODN versus imiquimod. Data are expressed as the geometric mean of the ratios calculated for each individual donor.PSME2, Proteasome activator complex subunit 2.


primates and that C- and D-ODN induce IFN-�2 secretion inmacaque PBMC cultures [28, 35, 36]. In vitro stimulation ofmacaque PBMCs (n�3) showed that the kinetics of inductionof IFN-�2 replicated that observed in humans, as imiquimodinduced earlier mRNA expression when compared with C- orD-ODN, which was elevated after 12 h in culture (Fig. 4A). Inaddition, as observed in human cells, the expression of mRNAinduced by D-ODN, but not C-ODN, remained elevated signifi-cantly for 24 h, leading to overall higher transcript levels (datanot shown) and increased protein expression [35]. In ma-caques, the repertoire of I-IFNs includes, in addition to IFN-�,six types of IFN-� (IFN-�1/13, -�2, -�6, -�8, -�14, and -�16),which have homology to human genes, and seven IFN genesthat have homology to IFN-�4, -�17, and -�21 (recentlytermed IFN-�23, -�24, -�25, -�26, -�27, -�28, and -�29 [9]). Toassess the complete I-IFN response in this species, a novel ar-ray of primer/probe sets specific for the mature coding se-

quence of each macaque IFN-� subtype was developed (un-published results) and used to test the mRNA levels in PBMCsfrom rhesus macaques (n�3), stimulated in vitro for 4 h or12 h. As shown in Fig. 4B, after 4 h in culture, cells stimulatedwith imiquimod showed increased levels of mRNA for IFN-�,as well as IFN-�1/13, -�2, -�6, and -�8, and to a lesser extent,IFN-�14, -�16, -�23, -�25, -�26, and -�28. Minimal (�10�) orundetectable, increased expression was evident in mRNA forIFN-�24, -�27, or -�29. By 12 h, only IFN-�, -�1, and -�8 re-mained increased significantly (fold increase �10 over base-line). In contrast, cells stimulated with D- or C-ODN showedlow levels of mRNA expression at 4 h, with predominant up-reguation of IFN-�1, -�6, and -�8, but by 12 h, increased levelsfor all I-IFN subtypes were evident. As observed with humanPBMCs, some of the subtypes showed detectable mRNA levelsat baseline (�14, �16, �27–29; Fig. 4C). Thus, this accountedfor the apparent lower change in transcript levels; however,

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Figure 4. I-IFN gene and ISG expression in rhesus macaque PBMCs stimulated with TLR agonists in vitro. PBMCs of three young adult rhesus ma-caques were stimulated with 0.3 �M CpG D- or C-ODN or 10 �g/ml imiquimod for 48 h. mRNA levels were assessed by qRT-PCR using rhesusmacaque-specific primer/probe sets. Fold increases were calculated over the expression levels of the individual genes in the unstimulated cells.Geomean of the individual values is shown. One of two independent experiments with similar results. (A) Fold increase in IFN-�2 mRNA over48 h of culture. (B) Fold increase in mRNA expression of individual I-IFN subtype genes at 4 h and 12 h. (C) Baseline levels of IFN-�2 expressionin unstimulated cells, normalized to the levels of 18S (HKG) of each sample (2–�Ct). Values are mean � sem of three animals. One of three ex-periments with similar results. (D) Proportional (percentage of total fold increase) expression of each I-IFN gene in the overall I-IFN responseduring 48 h of in vitro culture. (E) Expression of Mx1 and OAS1 in macaque PBMC.



these genes had similar absolute Ct values as the other sub-types, suggesting that they were similarly expressed upon TLRactivation. As with human PBMCs, TLR9 agonists induce acomparable I-IFN signature, as D- and C-ODN induced propor-tionally similar increases in I-IFN subtypes (Fig. 4D). Of note,for TLR7 and -9, the fold increase in up-regulation of IFN-�8was substantially higher than for the other genes. Lastly, theexpression of ISGs Mx1 and OAS1 correlated with the levels ofI-IFN induced by TLR7 and -9 activation (Fig. 4E).

Expression of I-IFN in the skin of rhesus macaquesClinical applications for TLR7 and -9 agonists include the treat-ment of skin cancer, including melanoma, cutaneous lymphoma,and basal cell carcinoma [37–39]. Their proposed mechanism ofaction is the induction of local IFN-�, as well as the promotion ofT cell responses to the tumor. In addition, our group has shownthat i.d. administration of D-ODN protects macaques against cuta-neous leishmaniasis, a disease whose outcome has been linked tothe production of IFNs [35].

To characterize the I-IFN response in skin, macaques (threeto four/group) were treated i.d. with TLR7 (CL097) or TLR9(C- or D-ODN) agonists or saline in three different areas ofthe upper chest and arm. Skin biopsies were collected fromthe injection sites at 6, 24, and 48 h postinoculation to assesslocal mRNA expression, as compared with that of untreatedskin collected prior to inoculation. For this study, we usedCL097 as the TLR7 agonist, a water-soluble resiquimod deriva-tive that induces IFN-� expression by PBMCs with similar ki-netics and subtype distribution as imiquimod but yields higherlevels of IFN-� in vitro (Supplemental Fig. 1).

Intradermally administered TLR9 and -7 agonists activatedsimilar local inflammatory responses, characterized by in-creased expression of IFN-�, CXCL10, IL-1�, and IL-8 in skin(Fig. 5A) but elicited markedly different levels of IFN-�. Skinbiopsies from macaques treated with CL097 showed a selectiveincrease in mRNA for IFN-�1/13, -�6, and -�8, which disap-peared by 48 h (Fig. 5B). In contrast, C- and D-ODN increasedthe mRNA levels of all I-IFN subtypes, and the expression wassustained for at least 48 h (Fig. 5B and D). As observed invitro, rhesus IFN-�8 was the gene that showed the more robustincrease in expression (Fig. 5B and E). The apparently weakerincrease in expression for IFN-�1/13 compared with the acti-vation in vitro likely reflects the higher baseline levels of thisIFN evident in vivo (Fig. 5C). Indeed, low levels of severalIFNs were evident in unstimulated skin (Fig. 5C). Importantly,despite D-ODN inducing ten- to 100-fold higher levels of I-IFNs than C-ODN, the relative increase of individual IFN sub-types resulted in a similar subtype distribution signature forboth TLR9 agonists (Fig. 5E). As observed in vitro, the in-crease in situ of Mx1 and OAS1 expression follows the samepattern as that of I-IFN (Fig. 5F). This indicates that very lowlevels of IFN are sufficient to activate this ISG response in vivo,although it is also possible that I-IFN-independent mechanismscontribute to ISG expression in skin [40]; therefore, dataabout Mx1 and OAS1 as biomarkers of IFN-� activity in vivoshould be interpreted with caution.

DISCUSSION

Synthetic TLR9 and TLR7 agonists are being tested in clinicaltrials to improve the efficacy of vaccines, reduce viral load, re-direct allergic responses, and treat cancer. For several of theseapplications, their mechanism of action is thought to belargely based on their induction of I-IFNs; however, whetherthese receptors induce similar IFN responses remained unre-solved. Despite the high homology of the IFN-� genes and theuse of a common receptor, several reports suggest that individ-ual IFN-� subtypes differ in their effects on virus replication orsusceptibility to diseases [41–43]. Interestingly, recent studiessuggest that specific combinations of ISGs may be particularlyeffective against particular viruses [44], raising the possibilitythat individual pattern recognition receptors may tailor theIFN response to fine-tune the response to virus or other stim-uli. Understanding the biological significance of individualIFN-� subtypes has been hampered by their common signalingpath and the high number of ISGs that they regulate.

In the present study, we developed tools to dissect the I-IFNresponse to TLR9 and -7. We show that TLR7 and -9 induceIFN-� and most subtypes of IFN-�, but the kinetic, quantity,and quality of the expression of the I-IFN genes induced varysignificantly, depending on the stimuli. TLR7 agonist imi-quimod induced preferentially IFN-�1/13, -�2, and -�14 orIFN-�8 and -�1/13 in PBMCs of humans or macaques, respec-tively. In contrast, the three types of CpG ODNs tested in-duced the transcription of all subtypes of I-IFN genes and de-spite marked differences in transcription levels and kinetics ofexpression, had proportional distributions of the individualIFN subtypes, which then translated into similar ISG signa-tures. The pattern of ISG trancription induced, whereaslargely overlapping, showed subtle but consistent differenceswhen cells were stimulated with TLR7 or TLR9 agonists, asCpG ODN (regardless of type) induced higher transcript levelsof CXCL10, GBP1, ATF5, MHC class II, and TNFSF10 andlower MET than the same cells stimulated with imiquimod.

In vitro and in vivo, the kinetics of the response were mark-edly different for TLR7 and -9. TLR7 agonists induced a rapidbut short-lived up-regulation of I-IFN mRNA, which peaked at2–4 h and then waned. The response to TLR9 agonists wasslower: The difference in kinetics was particularly pronouncedfor D-ODN, which induced a slower and more sustained geneexpression in vitro and in vivo, which correlated with elevatedlevels of protein and increased transcription of ISGs. C-ODNs,which share most structural elements with B-ODN, inducedthe I-IFN response hours earlier than D-ODN, and the levelsof IFN mRNA returned to baseline 10–12 h earlier as well.The profiles of transcription were consistent in the blood ofall of the donors in the study and consistent with previous ob-servations describing rapid but transient induction of IFN-�2transcription using B-ODN or TLR7 agonists (imiquimod, re-siquimod) compared with that elicited by D- or A-ODN(TLR9) [45–47], underscoring the impact of the type of ago-nist on the kinetic of the response. Of note, similar differencesin response kinetics were also described for viruses, suggestingthat these results are not an artifact as a result of the use ofsynthetic ligands [45–47]. Therefore, the magnitude and ki-


netics of in vitro expression of I-IFN genes seemed to be asso-ciated with the type of TLR agonist, whereas the type of recep-tor activated appeared to determine the subtype of IFNs thatare preferentially expressed.

The mechanisms underlying these differences in kinetics areunknown as yet. One possibility is that the difference in timingis rooted in the trafficking of the TLR agonists to the endo-somes, where they can activate the receptors: B-ODN and C-ODN do not have poly-G tails and thus, are not subject toHoogsteen base-pairing and the formation of G-tetrads. As aresult, they do not form aggregates and are thought to rapidlylocalize to late endosomes, where they engage TLR9 and in-duce NF-�B activation and to a lesser extent, I-IFNs. In con-

trast, D-ODNs have poly-G strands and self-complementary se-quences, allowing for the formation of tetrads. They are re-tained in early endosomes and mainly induce IFN genetranscription through the nuclear translocation of IRF7 [48–51]. Alternatively, the trafficking and processing of the recep-tors from the ER to the endolysosome compartment could bethe determinant factor in the delayed IFN response to D-ODN.To date, the precise mechanisms controlling TLR localizationin individual cell types remain poorly elucidated, as most stud-ies have been conducted in transfected human embryonic kid-ney 293 or RAW264 cells, which do not process TLR9 andhave poor responses to D-ODN [46, 52]. It is known thatTLR9 traffics linked to membrane protein Unc-93 homolog B1

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Figure 5. In situ I-IFN gene expression in the skin of rhesus macaques treated i.d. with TLR agonists. Macaques (n�3–4/group) were treated withCpG D- or C-ODN (0.3 mg/site), CL097 (30 �g/site), or saline at three different sites of the arm and chest area. Skin biopsies were collectedfrom the sites of injection at 0, 6, 24, and 48 h postinoculation. Total RNA was extracted, and transcript levels were measure by qRT-PCR usingmacaque I-IFN primer/probe sets. (A) In situ expression (fold increase) of IFN-�, CXCL10, IL-1�, and IL-8 at 6, 24, and 48 h. (B) Fold increasein expression of individual I-IFN subtype genes. (C) Baseline levels of IFN-�2 expression in unstimulated cells, normalized to the levels of GAPDH(HKG) of each sample (2–�Ct). Values are mean � sem of three to four animals. (D) Cumulative fold increase expression of all I-IFN genes (calcu-lated as the geomean of the sum of the AUC of the individual I-IFN genes for each macaque). (E) Proportional (percentage of total fold in-crease) expression of each I-IFN gene in the overall I-IFN response during the 48 h following injection. (F) ISG (Mx1 and OAS1) expression lev-els in skin at 24 h post-TLR agonist inoculation. For graphs A, B, and F, the fold increase in gene expression was calculated based on the tran-script levels measured in pretreated skin and shown as the geomean of the values of each individual monkey.



from the ER resident pool, through the Golgi network to theendolysosome compartment, where it is proteolytically acti-vated in a cathepsin-dependent manner to become functional[27, 46, 52–54]. Moreover, recent studies from Sasai et al. [27]suggest that to induce IFN production, after cleavage, TLR9traffics in complex with adaptor-protein 3 to a lysosome-re-lated organelle, where it recruits MyD88, TRAF3, and IRF7.Although TLR7 is thought to require similar proteolytic activa-tion, it is possible that it may not require the redirection tothe lysosomal compartment to bind ligands such as imi-quimod, and this may underlie the difference in the kineticsof IFN induction [46].

Our results show that TLR7 induces a more restricted IFNrepertoire than TLR9 in humans and macaques. Furthermore,preliminary data from our lab show that stimulation via TLR3or TLR4 results in different I-IFN subtype signatures thanthose induced by TLR7 or TLR9 (data not shown) . Previousstudies about the regulation of I-IFN transcription have under-scored the importance of transcription factors IRF3, IRF7, andIRF9 in the regulation of I-IFN expression [55–57]. IRF3 isconstitutively expressed in most cells, whereas IRF7 is constitu-tively expressed in pDC but can be up-regulated rapidly byother cells in response to I-IFNs. Once activated, IRF3 andIRF7 are phosphorylated, homo- or heterodimerized, andtranslocated to the nucleus, where they regulate IFN and IRFtranscription [58]. Studies in mice had suggested that IRF3can induce the transcription of IFN-� and IFN-�4 as a result ofits restricted DNA binding specificity, whereas IRF7 activates arobust response comprised of a broader spectrum on IFN iso-types [49]. Although similar observations have not been madein human cells, the imiquimod-mediated burst of a few sub-types of IFN-�, followed by immediate shutdown, could reflectan effect akin to early activation mediated by IRF3, which re-sults in immediate protein expression and accelerated turn-over, thereby decreasing the levels of IRF3 and IFN expressionsoon after stimulation [55]. In contrast, the broader and moresustained expression of I-IFNs induced by TLR9 agonistswould be akin to achieving sustained levels of IRF7. Prelimi-nary studies from our lab show that this is the case (Supple-mental Fig. 2), but whether the sustained mRNA levels of IRF7lead to a broad and prolonged I-IFN expression or the highlevels of I-IFNs lead to prolonged IRF7 expression remains un-clear. IRF9 has also been shown to play a dual role: a positiveregulator of the I-IFN feedback through the formation of het-erotrimeric transcriptional activator ISGF3, consisting of IRF9,STAT1, and STAT2, which induce IRF7, or a downmodulatorof the IFN response by associating with suppressor of cytokinesignaling 1. In agreement with the model proposed by Mai-wald et al. [59], our studies do suggest that increased expres-sion of IRF9 is associated with the shutdown of the IFN re-sponse. Of note, although our results suggest a two-phase ex-pression for most IFNs in human PBMCs, they do not agreewith those of Genin et al. [55, 56], who used Namalwa B lym-phoid cells stimulated with Sendai virus. Future studies willneed to assess whether the kinetics of expression are cell type-dependent. Our results also depart from those put forward bySzubin et al. [57], showing similar expression levels and kinet-ics for individual IFN-� subtypes regardless of the stimuli. It is

possible that the concentration of CpG ODN used in thatstudy, which was 15 times higher than in ours, may underliethese differences.

Despite the importance of the rhesus macaque model forthe study of a variety of diseases that occur in humans, theIFN response had not been well-described in this species. In arecent study, Easlick et al. [60] measured nine IFN subtypes inSIV-infected macaques, but to our knowledge, our study is thefirst to characterize in detail the kinetics and quality of theI-IFN response induced by TLR activation in vitro and in vivoin this species and contrast it with human data. Of note, al-though the kinetics of I-IFN induction by TLR7 and -9 agonistsreproduced that observed in human PBMCs, with TLR7 induc-ing a more rapid increase in IFN mRNA expression thanTLR9 agonists, the predominant IFN-� subtypes in rhesus wereIFN-�8 and -�1/13, underscoring the limitations of the ma-caque model for preclinical studies. In humans, IFN-�8 hasbeen described as potently inhibiting cell proliferation anddelaying G1-S transition, in addition to its ability to induce ap-optosis when combined with IFN-� in cancer models [41]. An-tiviral properties against HCV have also been attributed toIFN-�8, which would make it more effective than IFN-�1 inresponse to viral infections [42]. However, it is important tonote that the current nomenclature for the rhesus IFN genefamily is based on genetic and not functional homology, andnew and more detailed studies will be needed to understandwhether the differences observed in the IFN subtypes are clini-cally meaningful. Until then, the limitations of the macaquemodel should be considered when interpreting preclinicaldata.

Although assessing IFN production in whole PBMCs, insteadof isolated pDCs, allows for interactions between cell subsets, itdoes not necessarily reproduce the effects in vivo. Although alltreatments led to the expression of similar levels of proinflam-matory cytokines in the skin, i.d. administration of CL097(TLR7/8 agonist) induced only low levels of IFN-�8, -�6, -�27,and -�1 transcripts. Preliminary data from our lab show noincrease in IFN expression levels or pDC numbers in drainingLNs, suggesting that the lower levels of IFN-� transcripts ob-served in the skin of CL097-treated macaques are not a resultof rapid diffusion of the product away from the area of injec-tion or that they induce cellular migration of local pDCs tothe LNs. Detailed studies about the intracellular localization ofinjectable CL097 will be needed to determine whether thecompound is not reaching the receptors in skin.

As observed for PBMCs in vitro, the relative induction ofI-IFN subtypes was comparable between C- and D-ODNs in theskin of CpG ODN-treated macaques, despite differences in themagnitude of the IFN response between the two ODN types.This confirms that the type of receptor plays an important rolein determining the quality of the IFN response. The predomi-nance of IFN-�27 over -�1/13 or -�6 in skin, as compared withthe PBMC stimulated in vitro, suggests that the quality of theIFN response is also determined partly by the cell types acti-vated.

Over the past 15 years, our understanding of how the innateimmune system recognizes and responds to microbes, trauma,and cell death has grown considerably. Most intriguingly, a


host of receptors,which recognize nucleic acids, includingTLR3, -7, -8 and -9, retinoic acid-inducible gene 1, melanomadifferentiation-associated gene 5, and a variety of newly identi-fied DNA receptors in the cytoplasm, has been described [18].Most of these sensors signal via the recruitment of MyD88 toinduce the activation of NF-�B and IRF7-dependent IFNs. It istherefore possible to imagine two scenarios: one, possiblymore efficient, where a system of nucleic acid receptors is de-signed to detect different types of invading microbes but thenconverges into a common route of innate immune activationor “effector” route. Alternatively is a model where the individ-ual receptors share some steps in the signaling path but ulti-mately activate specific effector routes with subtle but impor-tant differences, thus optimizing the response to the type ofpathogen recognized. The apparent redundancy in IFNs ap-peared to support the first model; however, our studies aremore in line with the second, by suggesting that the quality ofthe IFN response to individual pathogens is shaped by the typeof receptor, its agonist (sequence and structure), and the in-tracellular space where the recognition takes place. This sug-gests that the TLR-IFN network may be more sophisticatedthan thought previously, and improved understanding of itsregulation may allow for better therapeutic strategies.

AUTHORSHIP

The laboratory of D.V. conducted all of the studies in humanand nonhuman primate PBMCs. M.P. designed and optimizedthe PCR array for human subtypes; performed in vitro culturesof human and macaque PBMCs, RNA extraction, and qRT-PCR in human PBMCs; and coordinated experiments, designof experiments, and interpretation of data. K.W.T. performedISG analysis from human and macaque samples. L.T.G. devel-oped the technique for isolating mRNA from the skin and per-formed ELISA to measure IFN protein levels. C.T. collabo-rated in optimization of human IFN qRT-PCR and the execu-tion of the kinetic experiments. The laboratory of R.L.R.developed the qRT-PCR assays and assessed the IFN-� subtypesin the macaques. L.M.S. developed and validated the IFN-�subtypes and IFN-� primer/probe sets. K.D.K. measured thelevels of IFN subtypes in macaque samples (in vitro and invivo). J.B. is the veterinary in charge of the macaques andhelped obtain the peripheral blood and skin biopsies.

ACKNOWLEDGMENTS

The authors thank Dr. Ray Donnelly, Dr. Howard Young, andClaire Grunes for reviewing the manuscript. Salary support forK.W.T. and L.T.G. was provided through ORISE as an inter-agency agreement between the US Department of Energy andthe US Food and Drug Administration. In addition, we thankDr. Phil Snoy, Lewis Shankle, and the Animal Care Facilitystaff for their expert care of the nonhuman primates includedin this study.

DISCLOSURES

The assertions herein are the private ones of the authors and are not to beconstrued as official or as reflecting the views of the FDA at large.

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KEY WORDS:interferon subtypes � CpG ODN � imiquimod � macaques � skin


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TLR9 and TLR7 agonists mediate distinct type I IFN responses in humans and nonhuman primates in vitro and in vivo