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The Immune Response Modifier and Toll-Like Receptor 7 Agonist S-27609 Selectively Induces IL-12 and TNF- Production in CD11c⁺CD11b⁺CD8^- Dendritic Cells ¹

Department of Pharmacology, 3 M Pharmaceuticals, St. Paul, MN 55144

	Abstract

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IL-12 and TNF- production by dendritic cells (DCs) is a critical step in the initiation of local inflammation and adaptive immune responses. We show in this study that a small molecule immune response modifier that is a Toll-like receptor 7 (TLR7) agonist induces IL-12 and TNF- production from murine CD11c⁺CD11b⁺CD8^- DCs, a subset not previously known for this activity. Stimulation of these DCs through TLR7 in vivo induces significant cytokine production even 12 h after initial stimulation, as well as migration of the DC into T cell zones of the lymphoid tissue. In contrast, stimulation through TLR4 and TLR9 induced IL-12 production predominantly from CD8⁺ DCs, consistent with previously published data. All TLR stimuli induced the increase in surface expression of the activation markers B7-1, B7-2, and class II in both CD8⁺ and CD8^- DCs, demonstrating that CD8⁺ DCs do respond to TLR7-mediated stimuli. To date this is the only known stimuli to induce preferential cytokine production from CD8^- DCs. Given the efficacy of TLR7 agonists as antiviral agents, the data collectively indicate that stimulation of CD8^- DCs through TLR7 most likely plays a role in the generation of antiviral immune responses.

	Introduction

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Dendritic cells (DCs)³ are generally agreed upon as the primary initiators of adaptive immune responses (1, 2). Upon activation by a variety of physical, chemical, and inflammatory mediators within tissues, DCs, such as Langerhans cells in the skin, migrate to lymphoid tissue and take up residence in T cell areas. In the process, they up-regulate many surface molecules, such as class I, class II, B7-1 and B7-2, and CD40, that enhance their ability to present Ag to both CD4⁺ and CD8⁺ T cells. In addition, the activation of DCs can induce their ability to produce a variety of cytokines, such as IL-12 and TNF-, that also contribute to T cell activation, proliferation, and differentiation into effectors (1, 2).

Different populations of DC have been described that differ both in their localization within lymphoid tissue as well as their propensity to produce different cytokines in response to various stimuli. In the mouse spleen, the majority of DCs are either CD11c⁺CD11b⁺CD8^- or CD11c⁺CD11b^low/-CD8⁺ (3, 4, 5, 6). Additionally, these DCs segregate into different physical compartments of the white pulp areas of the spleen. The CD11b⁺CD8^- DCs segregate predominantly into the marginal zones surrounding the white pulp (2, 4, 5, 7, 8, 9, 10), although after activation they appear to be able to migrate into the central T cell zone (9, 11). In contrast, CD11b^low/-CD8⁺ segregate to the T cell areas in both the spleen and lymph nodes (2, 4, 5, 7, 8, 9, 10). As one might expect of the DCs residing in the T cell zones of lymphoid tissue, these DCs have been shown to be able to effectively cross-present Ag to naive T cells in vivo (12, 13, 14, 15, 16). Consistent with this, ex vivo analysis of various populations of DCs to produce cytokines has demonstrated that the CD8⁺ DCs are the predominant producers of inflammatory mediators such as IL-12 and TNF- (17, 18, 19, 20, 21) that are known to play a role in T cell activation.

That being said, under the appropriate conditions, CD11b⁺CD8^- DCs are also capable of both producing IL-12 and stimulating T cell activation. For example, in vitro derived DCs from bone marrow cultured in GM-CSF are at least phenotypically CD11b⁺CD8^- DCs and are able to produce IL-12 to a variety of stimuli (22, 23, 24, 25). More importantly, CD11b⁺CD8^- DCs isolated from in vivo are also capable of at least some IL-12 production, again providing the appropriate activation stimuli were used (26, 27, 28). CD11b⁺CD8^- DCs are also capable of cross-priming a CD8⁺ T cell response to viral particles (29) or cell-associated Ag (14) as well as soluble Ag to CD4⁺ T cells (15, 30), demonstrating the inherent flexibility of the DC subpopulations to function in multiple roles.

The human Toll-like receptors (TLRs) have been identified as molecules important in DC activation. Named for their homology to a protein involved in the induction of development and immunity in Drosophila (31), the TLRs are now known to play a critical role in the initiation of mammalian immune responses by their recognition of various microbial and viral molecules. Agonists for TLRs include the inflammatory mediators LPS, peptidoglycan, and CpG for TLRs 4, 2, and 9, respectively (32, 33, 34). The immune response modifiers (IRM) are imidazoquinolines and also have significant immunomodulatory capabilities. IRMs such as imiquimod, resiquimod (R-848), and S-27609 are known to induce DC cytokine production and activation marker up-regulation (35, 36, 37), B cell activation (38, 39), and in particular can induce significant amounts of type 1 IFN in a number of species (40, 41, 42, 43, 44). As a result of these immunomodulatory capabilities, IRMs have been shown to mediate strong antiviral and antitumor responses (40, 45, 46, 47), and are currently used clinically for treatment of virally mediated diseases such as genital warts and herpes. Our studies as well as those of others have demonstrated that at least some IRMs are agonists for TLR7 in both humans and mice (48, 49). Additionally, we (unpublished data) and others (50) have shown that these IRMs are also agonists for human, but not mouse, TLR8.

Recent data have demonstrated the ability of TLR7 agonist IRMs to stimulate human DCs to differentially produce a variety of cytokines (48, 49, 51). In these studies, we define subsets of murine DCs capable of responding to a TLR7 agonist and demonstrate that the CD11b⁺CD8^-, but not CD11b^low/-CD8⁺, DCs produce IL-12 and TNF in response to TLR7 stimulation, despite the fact that both DCs increase activation marker expression. This stimulation of the myeloid DCs results in their migration from the marginal zones into the T cell zones within lymphoid tissue.

	Materials and Methods

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Mice

Female C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). All mice were maintained in our pathogen-free facility and used at 6–10 wk of age. All experiments were performed in accordance with 3M animal handling guidelines.

Antibodies

The following mAbs were purchased from BD PharMingen (San Diego, CA): 53-6.7 FITC (anti-CD8); M1/70 PerCP Cy5.5 (anti-CD11b); HL3 APC (anti-CD11c); C15.6 PE (anti-IL-12); MP6-XT22 PE (anti-TNF-); XMG1.2 PE (anti-IFN-). The 2.4G2 (anti-FcRII) was purified from supernatant grown in serum-free hybridoma medium (Sigma-Aldrich, St. Louis, MO).

IRMs

The IRM 1-(4-amino-2-methyl-1H-imidazo[4, 5-c]quinolin-1-yl)-2-methylpropan-2-ol hydrochloride (S-27609) was synthesized, as previously described (41). It was reconstituted in PBS at 10 mg/ml and diluted into PBS to 1 mg/ml for injection into mice i.v.

Human embryonic kidney (HEK) 293 transfection and IRM stimulation

HEK293 cells (passage 9) ATCC (American Type Culture Collection, Manassas, VA) CRL-1573 derived from human embryonal kidney and transformed with adenovirus 5 DNA were transfected in a 4:1 ratio with TLR7 (pRES expression vector; Clontech, Palo Alto, CA) or TLR9 (pMACSk^k expression vector; Miltenyi Biotec, Auburn, CA) and NF-B luciferase (Clontech). Cells were adhered to 24-well plates (Falcon 3047; BD Biosciences, Franklin Lakes, NJ) for 24 h at 37°C, 5%CO₂, then transiently transfected with TLR7 or TLR9 along with NF-B luciferase with Fugene 6 transfection reagent (Roche, Indianapolis, IN), following the manufacturer’s instructions. The plates were incubated for 24 h at 37°C, 5%CO₂ following tranfection and then stimulated with S-27609 at 30, 10, 3, and 1 µM and CpG 1006 at 30, 10, 3, and 1 µM. Cells were also incubated with DMSO alone as a vehicle negative control and with 1000 ng/ml of TNF as a positive control. The plates were then incubated an additional 24 h at 37°C, 5% CO₂. The luciferase signal was read in Optilux plates (Packard, Meriden, CT) using the Packard LucLite kit. The luminescence was measured on the Packard Topcount NXT (Packard), and measurement units of counts per second were obtained.

Mouse injections and DC isolation

Mice were injected i.v. with 200 µg of S-27609 (roughly 10 mg/kg). Blood was taken at various times by tail bleed, and the serum was assessed for the presence of cytokines by ELISA (BioSource International, Camarillo, CA). Alternatively, at various times after S-27609 injection, spleens were removed and collagenase digested, as previously described, to release the DCs (52). Briefly, spleens were minced with tweezers and incubated with 0.5 mg/ml collagenase D (Roche) in the presence of 1 µM GolgiPlug (BD Pharmigen) in 2 ml of chicks media (BioSource International) for 40 min at 37°C and 5% CO₂. A total of 2 ml of 0.1 M EDTA (Sigma-Aldrich) in Dulbecco’s PBS (BioSource International) was added after incubation at 37°C for 5 min. The spleen cells were washed with 0.5 mM EDTA in EHAA and resuspended in complete S-MEM medium (Biosource) (10% FCS (Atlas, Fort Collins, CO), 1% penicillin/streptomycin, 0.05 mM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x MEM nonessential amino acids). The cells were then incubated in the presence of 1 µM GolgiPlug for 2–3 h at 37°C to allow intracellular accumulation of cytokines. Surface Abs for flow cytometry were added to the cultures for the last half hour of the incubation. Cells were washed, resuspended in CytoFix (BD Pharmigen), incubated for 30 min at 4°C, and washed twice with PermWash (BD PharMingen). Cells were incubated with intracellular Abs in PermWash at 4°C for 45 min, washed with PermWash, and resuspended in FACS buffer (BD PharMingen). Samples were run on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences). At least 3000–5000 CD11c⁺ events were acquired to allow adequate assessment of the cytokine profiles of the DC subsets.

Immunohistochemistry

Eight hours after S-27609 injection i.v., spleens were taken and frozen in OCT mounting medium (Tissue Tek; Sakura, Tokyo, Japan). Sections (5–10 µM) were made on a cryostat (Leica, Deerfield, IL) of spleens from control and S-27609-treated mice. The sections were fixed in acetone for 5 min, dried, hydrated in PBS, and blocked with .5% goat and .5% donkey serum in PBS. The slides were then placed in 2% peroxide to neutralize endogenous peroxidase activity and then incubated with primary Ab (anti-CD11b or anti-CD11c) in blocking solution (DAKO, Carpinteria, CA). The slides were washed three times for 5 min each and incubated with a biotinylated anti-rat or anti-hamster secondary Ab, followed by a tertiary incubation with streptavidin (SA) HRP (DAKO). The slides were then developed with diaminobenzidine (DAKO), washed, and reincubated with peroxide. The slides were then treated with avidin, followed by biotin (DAKO), to block any remaining free SA biotin binding sites. The slides were then treated in a similar fashion for 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (DAKO) staining with anti-B220 biotin or CD8 biotin, followed by SA-alkaline phosphatase.

Cell isolations, ELISA, and quantitative reverse transcriptase PCR (QRT-PCR)

Cells from collagenase-digested spleen cells were stained with anti-B220 and anti-TCR FITC, then bound to anti-FITC beads and depleted of T and B cells over a magenetic bead column (Miltenyi Biotec). The remaining cells were then stained with anti-CD11c APC, CD11b PerCP, and CD8 PE. The Cd11c⁺CD8⁺ DCs and the CD11c⁺CD11b⁺ (CD8^-) DCs were then sorted to greater than 98% purity on a FACSVantage (BD PharMingen). The DCs were resuspended in complete medium containing GM-CSF (200 U/ml), IL-4 (100 U/ml), and IFN- (20 ng/ml) (26, 53) and stimulated for 18 h with a titration of S-27609 or CpG 1826. The supernatants were then analyzed for IL-12p70 by ELISA (Biosource).

Alternatively, total RNA was isolated from the given cell lines and isolated DC subtypes using the RNEasy kit (Qiagen, Valencia, CA). cDNA was prepared using the RETROScript First Strand synthesis kit (Ambion, Austin, TX), according to the manufacturer’s instructions. The obtained cDNA was diluted 1/10 with water, and 1 µl was used for amplification. Amplification was done using the SYBR Green PCR Master Mix Kit (Applied Biosystems, Warrington, U.K.) on a GeneAmp 5700 SDS. Invitrogen (Carlsbad, CA) synthesized PCR primers for TLR7; primer set 1, µ TLR7 sense (GTAAAT ATC CCA GAG GCC CAT GTG A), µ TLR7 antisense 272 (TCT GGA GAG ATG CTT GGT ATG TGG T), or µ TLR7 antisense 287 (GCC TAC GGA AGG AAT CTG GAG AGA); primer set 2; and GAPDH primers were purchased from Applied Biosystems. Relative expression was calculated using the Ct method by normalizing each sample to GAPDH.

	Results

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The IRM S-27609 is a TLR7 agonist and induces TNF- and IL-12 from CD11b⁺CD8^- myeloid DCs

The 3M compound S-27609 is an IRM (41, 43, 54, 55) and induces NF-B nuclear localization by signaling through TLR7 (Fig. 1), similar to other 3 M antiviral compounds such as imiquimod and resiquimod (R848) (48, 49). Also similar to R848 (48, 49), TLR7-mediated stimulation by S-27609 in mice results in the production of IFN-, TNF-, and IL-12 that can be detected in the serum (Fig. 2). It has been recently demonstrated that IFN- is produced by the plasmacytoid DCs (49, 56), which in the mouse are CD45A, Ly-6C, and/or GR-1⁺, B220⁺, and CD11c^low (49, 57, 58, 59). Similar results were seen in our studies (data not shown), but we were interested to know which of the other cell populations might be responsible for the production of IL-12 and TNF-. Previous data from both mouse and human cells have demonstrated that IRMs act primarily on APCs (35, 36, 37, 38, 60, 61). As a result, we specifically examined various populations of APCs by flow cytometry to assess their responses to IRM stimulation in vivo.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. The IRM S-27609 is a TLR7 agonist. HEK293 cells were transiently transfected with constructs for TLR7 or TLR9 and cotransfected with NF-B luciferase expression construct, as described in Materials and Methods. The transfectants were incubated with S-27609, CpG 1006, DMSO only (vehicle negative control), or 100 ng/ml TNF (positive control for NF-B-induced expression). Twenty-four hours later, the cells were analyzed for luciferase activity as a measure of the level of NF-B signal induced by the IRMs. The data are expressed as counts per second.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. IRM induces cytokine production detectable in the serum. Mice were injected with 200 µg of S-27609 i.v., and the serum was taken at the indicated time points. Serum was analyzed for the presence of TNF- and IL-12 by ELISA (expressed in picograms per milliliter) and for IFN- by bioassay (expressed as units per milliliter). Each data point is an average from three mice per treatment group per time point.

View larger version (44K): [in this window] [in a new window]   FIGURE 3. IRM induces IL-12 and TNF- production from CD11b+CD8- DCs. Mice were injected with 200 µg S-27609 i.v. 2 h later, spleens were removed, and collagenase digested to release the DCs. The cells were then incubated at 3–4 h in the presence of brefeldin A and then stained and analyzed by FACS intracellular cytokine production as described in Materials and Methods. Either all CD11C+ cells (gate R2) (A) or CD11b+CD11c- cells (gate R3) (B) were plotted for their CD8 expression and analyzed for IL-12, TNF-, and IFN- production. The data presented are representative of six separate experiments.

Because detectable levels of IL-12 and TNF- in the serum were transient, we speculated that the ability of the DCs to produce cytokine would be short-lived as well. Mice were injected i.v. with IRM and, at various times after, spleen DCs were assessed for their production of cytokines as before. Again we were surprised to see that both IL-12 and TNF- were produced by a significant percentage of CD11b⁺CD11c⁺ and CD11b⁺CD11c^- cells, respectively, even out to 12–24 h after initial IRM treatment (Fig. 4), well past when the drug itself is detectable in the serum based on pharmacokinetic studies (data not shown). IL-12 (Fig. 4) and TNF- (not shown) production from the CD11b⁺CD8^- DCs usually declined to just above background levels between 12 and 24 h, but TNF- production was easily detectable through 24 h in the monocyte/macrophage cells. Thus, the DCs within the lymphoid tissue retain their ability to produce cytokine for longer periods than the serum levels would necessarily indicate. As before, CD11b^-CD8⁺ DCs did not produce significant levels of either TNF- or IL-12 at any time point (Fig. 4).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. IL-12 and TNF- production is sustained through 12–24 h after IRM-mediated TLR7 stimulation. Mice were injected with 200 µg S-27609 i.v. and, at the times indicated, spleens were removed and collagenase digested and stained for intracellular cytokine production of DCs (A) or monocytes/macrophages (B), as in Fig. 3 and as described in Materials and Methods. The events shown in A were gated as in the R2 gate, and B were gated as in the R3 gate, from Fig. 3. Numbers on the dot plots indicate the percentage of cells in the lower right quadrant of the total cells shown. The data are representative of three independent experiments.

Other groups have assessed the ability of various DC subpopulations to produce cytokines and have determined that the CD11b^-CD8⁺ DCs are the predominant producers of IL-12 (17, 18, 26, 62). However, the stimulation used to elicit the DC response in these studies was with TLR agonists other than for TLR7. We therefore assessed the response of both DC subsets in our model system to stimulation with LPS or CpG, which are TLR4 and TLR9 agonists, respectively. Mice were treated in vivo with each agonist, followed by the removal and intracellular staining of splenic DCs at various time points, as before. In contrast to the IRM, the other TLR agonists stimulated IL-12 production from both CD11b^-CD8⁺ and CD11b⁺CD8^- DCs (Fig. 5A). As a percentage of the total cells in the given subset, CD8⁺ DCs produced the most IL-12 (Fig. 5B). LPS induced the greatest IL-12 production 1 h after stimulation, while the IRM and CpG induced increased IL-12 production through 8–12 h (Figs. 5B and 4A, and data not shown). TLR2 and TLR3 agonists (peptidoglycan and poly(I:C), respectively) also demonstrated a more selective activation of the CD11b^-CD8⁺ DCs as well, although they both induced an overall lower amount of cytokine than LPS and CpG (data not shown). Consistent with the in vivo data, in vitro stimulated CD8^- DCs produced IL-12 p70 in response to S-27609, while CD8⁺ DCs produced IL-12 p70 in response to CpG (Fig. 6). Thus, IL-12 can be induced, both p40/70 in vivo and p70 in vitro, by S-27609 from the CD8^- DC subset.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. IRMs are unique in stimulating IL-12 production from CD11b+CD8- DCs. Mice were injected with either 200 µg S-27609, 50 µg CpG 1826, or 30 µg LPS i.v. 1 h later spleens were removed, treated, and stained as in Figs. 3 and 4. A, The dot plots shown are gated on all CD11c+ events as in gate R2 of Fig. 3. The TLR that each reagent stimulates is given in parentheses. B, Spleens from the mice in A were removed at various time points and treated as in A. The percentage of cytokine-producing DCs was calculated by gating on either CD8+CD11c+ or CD11b+CD11c+ DCs. The percentage of IL-12-producing cells of each of those populations was then identified, and the data are expressed as the percentage of cytokine-producing DC within either the CD8- (left graph) or CD8+ (right graph) DC subsets. Results are representative of three independent experiments.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. The S-27609 stimulates IL-12p70 production from DCs in vitro. CD8+ and CD8- DC subsets were sorted to >98% purity, as described in Materials and Methods, cultured at 1 x 105 cells/ml, and stimulated with either 1 µM S-27609 (solid bars) or 1 µg/ml CpG 1826 (hatched bars) overnight. Supernatants were analyzed by ELISA for the presence of IL-12p70 (Biosource). The data are expressed as picograms per milliliter and are representative of two experiments performed.

View larger version (45K): [in this window] [in a new window]   FIGURE 7. IRM induces activation marker up-regulation in both CD8+ and CD8- DC subsets. Cells from Fig. 5 were stained with Abs to B7-2, CD40, and class II to assess levels of surface expression. A, Surface expression of the given markers on either the CD11b-CD8+ (left) or CD11b+CD8- (right) DCs 8 h after initial i.v. injection. Open histograms indicate staining of the given marker on control DCs, and the filled histogram indicates staining on the DC from the treated mice. B, Time course of B7-2 (top) and class II (bottom) expression comparing CD11b-CD8+ (left) and CD11b+CD8- (right) from CpG-treated (diamonds) and S-27609-treated (squares) mice. The mean fluorescence intensities (MFI) for each marker were calculated from histograms shown in A. The 0 time point indicates the MFI of the control mice. Results are representative of three experiments performed.

View larger version (21K): [in this window] [in a new window]   FIGURE 8. Both CD8+ and CD8- DCs express TLR7, but CD8- DCs express 3- to 5-fold more. DCs were isolated and RNA purified, as described in Materials and Methods. Two primer sets for analysis of TLR7 were developed and used in quantitative RT-PCR analysis of the isolated subsets. The RMAS and EL4 cell lines were used as negative controls. The data are expressed as the expression of TLR7 in the given cell type relative to GAPDH within that cell type. This experiment is representative of two performed.

Previous data have shown that upon activation, DCs migrate into the T cell zones of the lymphoid tissue (9, 11, 37). To insure that the IRM treatment was indeed inducing this aspect of DC activation, we analyzed spleen sections from mice treated with the IRM by immunohistochemistry staining for the locations of CD11b⁺ and CD11c⁺ cells. Naive mice demonstrated strong staining of DCs both in the marginal zones in which the CD11b⁺CD8^- DCs reside, as well as in the T cell zones in which the CD11b^-CD8⁺ DCs reside (Fig. 9, CD11c/B220). After treatment with the IRM, DC staining from the marginal zones was dramatically decreased as the staining of the DCs in the T cell zones was increased (Fig. 9, CD11c/B220). In addition, although CD11b staining was virtually absent from the T cell zones of the naive mice, significant CD11b staining within the T cell zones was seen after treatment with IRM (Fig. 9, CD11b/CD8). Many of these cells costained with anti-CD11c (data not shown), indicating they were indeed DCs. These data confirm that the CD11b⁺CD8^- DCs from the marginal zones (CD11b⁺) were induced to migrate into the T cell zones after stimulation with a TLR7 agonist. Therefore, TLR7 agonists can induce all of the features of DC activation within the CD11b⁺CD8^- DC subset; cytokine production, activation marker expression, and migration.

View larger version (133K): [in this window] [in a new window]   FIGURE 9. TLR7 stimulation in vivo induces CD11b+CD8- DC migration from the marginal zones into the T cell zones in the spleen. Mice were injected with 200 µg S-27609 i.v., and 6 h later, the spleens were removed and frozen in mounting medium. Serial sections (5 µm) were made on a cryostat and stained by immunohistochemistry, as described in Materials and Methods, for CD11b+ cells (right) or CD11c+ cells (left). The slides were developed with diaminobenzidine, so cells staining with the indicated Ab appear brown, an example of which is indicated with the arrowhead in the S-27609 treatment slide. The slides were also stained for B220+ or CD8+ cells and developed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (blue) to identify the B and T cell zones, respectively. The letters indicate the various regions of the white pulp. T, T cell zones; B, B cell zone; MZ, marginal zone.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It is interesting to note that others have reported a connection between the CD11b⁺CD8^- DC subset and the activation of a Th2-like T cell response (17, 27, 64, 65, 66). Those studies showed that the use of CD11b⁺CD8^- DCs to prime T cell responses produced predominantly Th2-like responses. In contrast, IRMs have demonstrated the ability to skew away from Th2-like and toward Th1-like responses (36, 67). In those studies, IRM treatment along with antigenic challenge in the presence or absence of Alum both inhibited the production of IgE and dramatically augmented the production of IgG2a. Th1 cytokines such as TNF- and IFN- were also increased in the presence of IRMs, as well as IL-12, as we have shown in this work. Previous results from other groups have demonstrated that CD8^- DCs could induce Th1-like T cell responses provided the appropriate activation stimulus was provided (14, 24, 25, 29), and our studies demonstrate that this may be able to be achieved by activation of the DCs through TLR7.

A natural ligand/agonist for TLR7 is currently not known. We speculate, however, that it will be found to be some component of a virus or a viral infection, given the strong IFN response of both mouse and human to TLR7 agonists (40, 41, 42, 43, 44, 48, 49). It has recently been demonstrated that the CD11b⁺CD8^- DC subset in the spleen is capable of the capture and presentation of viral particle-derived Ags (29). This is also the main DC subset to present Ag to CD4 T cells in the draining lymphoid tissue in response to s.c. Ag challenge (30). Given this, as well as the fact that the TLR7 agonist Aldara/imiquimod is already proven to be successful as an antiviral agent in treating human papillomavirus-induced genital warts in the clinic (40, 45, 46, 47, 68, 69), the results we have demonstrated in this work further supports the speculation that TLR7 and CD11b⁺CD8^- DCs play an important role in clearing viral infections. Given our demonstration of the importance of CD8^- DCs in response to TLR7 agonistic IRMs, ongoing work will determine how this DC subset contributes to the initiation or propagation of antiviral and other adaptive immune responses.

	Acknowledgments

 
Thanks to Dr. Mark Tomai and Dr. Richard Miller for helpful comments on the manuscript. Thanks also to Sean McGurran for assistance in in vivo experiments.

	Footnotes

 
¹ C.L.D. and T.R.R. contributed equally to the manuscript.

² Address correspondence and reprint requests to Dr. Ross M. Kedl, 3 M Pharmaceuticals, 3 M Center Building 270-2S-06, St. Paul, MN 55144. E-mail address: rmkedl{at}mmm.com

³ Abbreviations used in this paper: DC, dendritic cell; HEK, human embryonic kidney; IRM, immune response modifier; QRT-PCR, quantitative reverse transcriptase PCR; SA, streptavidin; TLR, Toll-like receptor.

Received for publication October 8, 2002. Accepted for publication May 29, 2003.

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