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Human Dendritic Cells Stimulated via TLR7 and/or TLR8 Induce the Sequential Production of Il-10, IFN-γ, and IL-17A by Naive CD4 + T Cells This information is current as of September 23, 2021. Vincent Lombardi, Laurence Van Overtvelt, Stéphane Horiot and Philippe Moingeon J Immunol 2009; 182:3372-3379; ; doi: 10.4049/jimmunol.0801969 http://www.jimmunol.org/content/182/6/3372 Downloaded from

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human Dendritic Cells Stimulated via TLR7 and/or TLR8 Induce the Sequential Production of Il-10, IFN-␥, and IL-17A by Naive CD4؉ T Cells

Vincent Lombardi, Laurence Van Overtvelt, Ste´phane Horiot, and Philippe Moingeon1

Depending upon which TLRs are triggered, dendritic cells (DCs) may orient the differentiation of naive CD4؉ T cells toward either Th1, Th2, regulatory T cells, or the recently defined Th17 lineage. In this study, we report that a dual stimulation of TLR4 and TLR7/8 with LPS plus R848 leads human monocyte-derived DCs (MoDCs) to produce multiple pro- and anti-inflammatory cytokines, including IL-10, IL-12, and IL-23. Surprisingly, a significant variability in the up-regulation of these cytokines is observed in DCs obtained from various healthy donors, with approximately one of three being “high responders.” High responding MoDCs stimulated via TLR4 and TLR7/8 induce naive allogeneic CD4؉ T cell to secrete sequentially IL-10 and IFN-␥, and Downloaded from eventually IL-17A, whereas low responding MoDCs only stimulate IFN-␥ production. Both TLR7 and TLR8 play a central role in this phenomenon: TLR4 triggering with LPS up-regulates TLR7 expression on human MoDCs from high responders, silencing of either TLR7 or TLR8 mRNAs inhibits cytokine production in LPS plus R848-treated MoDCs, and plasmacytoid DCs constitutively expressing high levels of TLR7 induce the production of IL-10, IFN-␥, and IL-17A by naive T cells when stimulated with R848 alone. Collectively, our results illustrate the synergy between TLR4 and TLR7/8 in controlling the sequential production of ؉

regulatory and proinﬂammatory cytokines by naive CD4 T cells. The observed polymorphism in DC responses to such TLR- http://www.jimmunol.org/ mediated stimuli could explain differences in the susceptibility to infectious pathogens or autoimmune diseases within the human population. The Journal of Immunology, 2009, 182: 3372–3379.

ollowing engagement with conserved microbial motifs (1– (5–9, 15–18). Th17 cells contribute to defense mechanisms against 3), TLRs play a critical role in inducing appropriate im- extracellular infectious pathogens but may also cause autoimmune mune responses against potential pathogenic agents (4). diseases (19–23). Immune responses to a pathogen or a danger F 2 Most particularly, signals provided to dendritic cells (DCs) signal are thus the end result of a balance between effector (proin- through selected TLRs are known to impact dramatically the cy- flammatory) and regulatory mechanisms involving those various ϩ tokine milieu in which the Ag is presented, thus leading to distinct CD4 T cell subsets at varying levels in the course of the response. by guest on September 23, 2021 patterns of naive CD4ϩ T cell differentiation (5–9). Th1 cells are In the present study, we document that both human monocyte- induced by IL-12-producing APCs, produce IFN-␥, and protect derived (MoDCs) and plasmacytoid DCs (pDCs) produce high lev- against intracellular bacteria or viruses. Chronic stimulation of Th1 els of IL-10, IL-12, and IL-23, albeit with unexpected variability cells may lead to autoimmunity (10). Th2 cells are elicited by depending upon individuals, when receiving a strong signal via IL-4-secreting APCs and produce IL-4, IL-5, and IL-13 cytokines. TLR7 and/or TLR8. High responding DCs support a sequential As such, they represent an effective defense mechanism against differentiation of naive CD4ϩ T cells into IL-10, IFN-␥, and IL- parasites, but are also associated with type I allergic inflammation 17-producing cells, likely to mediate both regulatory and proin- (11). CD4ϩ regulatory T cells (Tregs) capable of suppressing both flammatory functional programs, respectively. Th1 and Th2 immune responses are induced by DCs producing IL-10 and/or TGF-␤. Naturally occurring Tregs express the tran- Materials and Methods ␤ scription factor Foxp3 and produce IL-10 and/or TGF- , whereas Cells, reagents, and DC/T cocultures Th3 and Tr1 cells produce either TGF-␤ or IL-10, respectively (12–14). Recently, several studies established that DCs producing Heparinized blood from healthy volunteers (obtained from Etablissement Français du Sang) was centrifuged over a Ficoll-Paque plus gradient (GE IL-1␤, IL-6, IL-23, and/or TGF-␤ induce the differentiation of a 8 ϩ Healthcare) to isolate PBMCs. To generate MoDCs, 10 cells were cul- new population of IL-17A-secreting CD4 Th cells termed Th17 tured at 37°C in 5% CO2 in a 75-ml plastic flask in 25 ml of culture medium (RPMI 1640 supplemented with 2 mM L-glutamine, 20 ␮g/ml gentamicin, 50 ␮M 2-ME, 1% nonessential amino acids (all obtained from Invitrogen) and 10% FBS (Gentaur)). After 2 h, nonadherent cells were Research and Development, Stallerge`nes SA, France removed, and adherent cells were further cultivated for 7 days in presence Received for publication June 17, 2008. Accepted for publication January 6, 2009. of recombinant human GM-CSF and IL-4, 80 and 25 ng/ml, respectively The costs of publication of this article were defrayed in part by the payment of page (Gentaur). After 7 days at 37°C, a pure population of DCs was obtained, ϩ charges. This article must therefore be hereby marked advertisement in accordance with more than 95% CD1a cells detected by flow cytometry using a with 18 U.S.C. Section 1734 solely to indicate this fact. FC500 cytometer and CXP analysis software (Beckman Coulter). To test 6 1 Address correspondence and reprint requests to Dr. Phillipe Moingeon, Research the effect of TLR ligands on DCs, 10 MoDCs were plated in a 24-well and Development, Stallergenes S.A., 6 Rue Alexis de Tocqueville, Antony Cedex plate in 1 ml of culture medium in presence of either medium, highly 92183, France. E-mail address: [email protected] purified LPS from Escherichia coli (1 ␮g/ml; InvivoGen), Resiquimod ␮ 2 Abbreviations used in this paper: DC, dendritic cell; CBA, cytometric bead array; (R848, 1 rusha g/ml; InvivoGen) or a combination of LPS plus R848. After 24 h at 37°C and 5% CO2, MoDCs were lysed to isolate total RNA MoDC, monocyte-derived dendritic cell; pDC, plasmacytoid dendritic cell; Treg, reg- ϩ ulatory T cell. or washed and cultured in a 24-well plate with allogeneic CD4 naive T cells at a 1:10 DC/T ratio in 1 ml of culture medium. Naive CD4ϩ T cells ϩ Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 were isolated from PBMCs by negative selection using a Dynal CD4

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801969 The Journal of Immunology 3373 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. LPS plus R848-treated MoDCs trigger a sequential differentiation of naive CD4ϩ T cells toward IL-10, IFN-␥, and IL-17-producing T cells. MoDCs from nine healthy donors were treated for 24 h with either medium alone, LPS (1 ␮g/ml), R848 (1 ␮g/ml), or LPS plus R848, then washed and cultured with allogeneic naive CD4ϩ T cells. A, After 1, 3, and 6 days of culture, gene expression was quantified by real-time PCR. None of the cytokine genes was up-regulated in culture conditions lacking T lymphocytes. B, At day 3, 5, and 7, cells were restimulated for 4 h with a plate-coated anti-CD3 Ab in presence of Brefeldin A. Cells were surface stained with anti-CD4 and anti-CD25 Abs, fixed, and permeabilized to allow intracellular detection of IFN-␥, IL-10, and IL-17 by cytofluorometry. Data are displayed as dot plot histograms obtained after gating on CD4ϩ T cells and are representative of three independent experiments. 3374 TLR7/8-STIMULATED DCs INDUCE COMPLEX T CELL DIFFERENTIATION

FIGURE 2. A combination of LPS plus R848 up-regulates cytokine production by MoDCs. Human MoDCs obtained from 26 healthy donors were cultured with either medium, ultra-pure LPS (1 ␮g/ml), R848 (1 ␮g/ml), or LPS Downloaded from plus R848. A, After 24 h, total RNA was isolated and IL-12, IL-23, IL-6, IL-1␤, TNF-␣, IL-10, IL-4, and TGF-␤ gene expression was evaluated by quantitative real-time PCR. B, Cytokine production was measured in culture supernatants by CBA (IL-12p70, IL-6, IL-1␤, and IL-10) http://www.jimmunol.org/ and ELISA (IL-23). by guest on September 23, 2021

isolation kit (Invitrogen) according to the manufacturer’s instructions. Such quently washed and double-stained with either PE-labeled anti-IL-10 (BD naive CD4ϩ T cells were confirmed to be Ͼ95% pure based on CD3, CD4, Bioscience) or anti-IL-17A (eBioscience) Abs, as well as a FITC-conju- and CD45RA expression evaluated by flow cytometry. Neutralizing Abs gated anti-IFN-␥ Ab (BD Bioscience). Samples were analyzed on a FC500 specific for IL-10 (10 ␮g/ml; R&D Systems), IL-12 (10 ␮g/ml; R&D Sys- flow cytometer (Beckman Coulter). tems) and IL-23 (1 ␮g/ml; eBioscience) or an isotype matched control Ab (mouse IgG1, 1 ␮g/ml; BD Bioscience) were added to the cultures. To test the effect of R848-treated pDCs on T cell polarization, pDCs were sorted RNA isolation and quantitative real-time PCR analysis using a Diamond plasmacytoid dendritic cell isolation kit (Miltenyi Bio- tech) per the manufacturer’s instructions and confirmed to be Ͼ99% pure, Total RNA was extracted from DCs or T cells using a RNeasy mini kit based on BDCA2 and CD123 expression assessed by flow cytometry. (Qiagen), and cDNAs were synthesized using TaqMan reverse tran- pDCs were stimulated for 24 h with R848 (1 ␮g/ml), washed, and cultured scription reagents (Applied Biosystems) as per the manufacturer’s in- ϩ structions. mRNA expression was evaluated by quantitative PCR on a for up to 6 days with purified naive CD4 T cells at a 1:10 pDC/T ratio. 7300 real-time PCR system (Applied Biosystems) with predesigned Measurement of cytokine production Taqman gene expression assays and reagents, according to the manufacturer’s instructions. The expression of the following genes was assessed The production of IL-23 and IL-17A cytokines (eBioscience and R&D in MoDCs: IL-12p35 (Hs00168405_m1), IL-12p40 (Hs00233688_m1), IL- Systems, respectively) was evaluated by ELISA according to the manu- 23p19 (Hs00372324_m1), IL-1␤ (Hs00174097_m1), IL-6 (Hs00174131_ facturer’s instructions. Production of IL-1␤, IL-6, IL-10, TNF-␣, and IL- m1), TNF-␣ (Hs00174128_m1), and IL-4 (Hs00174122_m1). To monitor T 12p70 by MoDCs and IL-10 and IFN-␥ by CD4ϩ T cells were measured cell polarization, the expression of the following genes was evaluated: Tbet using the Flex Set cytometric beads array (CBA) (BD Bioscience), accord- (Hs00203436_m1), GATA-3 (Hs00231122_m1), Fox p3 (Hs00203958_m1), ing to the manufacturer’s instructions. Analysis of intracellular cytokine RORc2 (Hs01076112_m1), IFN-␥ (⌯s00174143_m1), IL-4 (Hs00174122_ levels was performed on naive CD4ϩ T cells cultured with MoDCs pre- m1), IL-10 (Hs00174086_m1), TGF-␤ (Hs00171257_m1), and IL-17A treated with TLR ligands. CD4ϩ T cells were recovered and restimulated (Hs00174383_m1). Data were interpreted, for each target gene monitored in with plate-bound anti-CD3 (1 ␮g/ml X35; Beckman Coulter) for4hin MoDCs or T cells, in comparison with endogenous ␤-actin (Hs99999903_m1) presence of GolgiPlug (BD Bioscience). Cells were surface-stained with as a control. RNA 18S (Hs99999901_s1) was used as a control for gene ex- anti-CD4 and anti-CD25 Abs (Beckman Coulter), then fixed and perme- pression analysis in pDCs. The relative amount of target genes in each sample abilized using the Intraprep reagent (Beckman Coulter). Cells were subse- was calculated in comparison with the calibrator sample using the ⌬⌬Ct The Journal of Immunology 3375 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Involvement of IL-10, IL-12, and IL-23 produced by LPS plus R848-treated MoDCs in the sequential polarization of naive CD4ϩ T cells. A, IL-10, IL-12p35, IL-12p40, and IL-23 gene expression by MoDCs treated with LPS plus R848 (1 ␮g/ml) was evaluated by real-time PCR, and correlated with IL-10, IFN-␥, and IL-17 gene expression by CD4ϩ T cells after 1, 3, and 6 days of coculture, respectively. Results from nine healthy donors are shown, distinguishing high (open triangles) vs low (black dots) responders. B, Neutralizing Abs specific for IL-10, IL-12, IL-23, or corresponding control isotypes (mouse IgG1) were added at the beginning of cocultures between LPS plus R848-treated MoDC and purified naive allogeneic CD4ϩ T lymphocytes. At day 5, total RNA was extracted and mRNA expression was quantified by real-time PCR. Data shown are the mean of four independent experiments Ϯ SEM. method. The magnitude of gene induction was calculated using the formula Results Ϫ⌬⌬Ct ϭ Ϫ(⌬Ct for unstimulated cells Ϫ⌬Ct for stimulated cells) 2 2 . Polarization of naive CD4ϩ T cells induced by MoDCs treated RNA interference with LPS plus R848 To inhibit TLR7 and/or TLR8 expression, Silencer Select Pre-designed We assessed the impact of DCs treated with LPS with or without siRNAs (Applied Biosystems) specific for TLR7 or TLR8 were used. In R848 on Th cell polarization. MoDCs were treated for 24 h with 5 ␮ each experiment, two 10 MoDCs were plated in 24-well plates in 400 l either medium, LPS, R848, or LPS plus R848, then washed and of growth medium containing 10% FBS without antibiotic. Lipofectamine ϩ 2000 (1 ␮l/well; Invitrogen) was first diluted in 50 ␮l of Opti-MEM (In- cultured with purified allogeneic naive CD4 T cells. After 1, 3, vitrogen) for 5 min before mixing with an equal volume of Opti-MEM and 6 days, the expression of genes encoding for specific cytokines containing 20 pmol of siRNA. After 20 min, 100 ␮l of the Lipofectamine/ and transcription factors associated with either Th1, Th2, Treg, or siRNA mix were added to the cells to perform siRNA transfection. Fresh Th17 differentiation was evaluated by quantitative PCR. As shown growth medium was added 4 h after transfection. Cells were cultured for ϩ 36 h at 37°C to obtain optimal silencing of targeted genes. The efficacy of in Fig. 1A, two distinct patterns of naive CD4 T cell differentiation gene silencing was tested by real-time PCR using siRNA control and were observed after coculture with LPS plus R848-treated MoDCs. In siRNA GAPDH as negative and positive controls, respectively. three of nine donors, a strong (i.e., at least 50-fold) induction of IL-10, IFN-␥, and IL-17A genes was detected after 1, 3, and 6 days, respec- Statistical analysis tively, of coculture (Fig. 1A, open triangles). This pattern of cytokine Levels of cytokine produced were compared using a Student’s t test. A gene expression was not observed in T cells cocultured with MoDCs Ͻ value of p 0.05 was considered statistically significant. The multiple treated with LPS or R848 alone (Fig. 1A). In cocultures made between regression correlation coefficient R2 was calculated to estimate the linear relationship between cytokine gene expression by MoDCs and cytokine MoDCs/T cells from most donors, only IFN-␥ gene expression was gene expression by CD4ϩ T cells. up-regulated to a lower level after 3 days (Fig. 1A, black dots). In 3376 TLR7/8-STIMULATED DCs INDUCE COMPLEX T CELL DIFFERENTIATION Downloaded from

FIGURE 4. Levels of TLR expression correlate with MoDCs responsiveness. A, TLR gene expression was evaluated by real-time PCR in ﬁve different

donors after treatment of MoDCs with LPS and/or R848. B, Basal expression http://www.jimmunol.org/ of TLR4, 7, and 8 genes was determined in unstimulated MoDCs. Results are displayed for high (open triangles) and low (black dots) responding donors. All PCR data shown are normalized to amounts of ␤-actin mRNA, with values for negative controls (i.e., unstimulated DCs in A, DCs from donor with lowest level of TLR expression in B) arbitrarily set to 1.

contrast to such low responding patients, high responders are thus defined based on the capacity of their MoDCs to induce a strong and by guest on September 23, 2021 ϩ FIGURE 5. Role of TLR7 and TLR8 in the up-regulation of cytokine sequential expression of IL-10/IFN-␥/IL-17A genes by naive CD4 T production by MoDCs. MoDCs were transfected with siRNAs specific for cells, after LPS plus R848 stimulation. ϩ either TLR7 or TLR8, used alone or in combination, or with a control Using intracellular cytokine staining, IL-10-secreting CD4 T irrelevant siRNA. Thirty-six hours after transfection, cells were activated cells were detected at day 3 only in cocultures obtained with high with LPS plus R848 for 24 h. The efficacy of TLR7 and TLR8 gene si- responding MoDCs treated with LPS plus R848 (Fig. 1B). IFN-␥ lencing was evaluated by real-time PCR. A, Data shown are the mean of production was up-regulated in T cells with a peak at day 5 three independent experiments Ϯ SEM. Supernatants were harvested and whereas IL-10 was no longer detected in culture supernatants (Fig. cytokine production was evaluated by CBA for IL-10 and IL12p70, and by 1B). Multiple labeling experiments established that IL-10 and IFN- ELISA for IL-23. Results are the mean of three independent experiments Ϯ .p Ͻ 0.02 ,ءء ;p Ͻ 0.05 ,ء ,secreting cells represent two distinct populations (Fig. 1B), and SEM. B-␥ that IL-10-producing T cells are CD25 negative (data not shown). No significant differences were observed regarding Foxp3 expres- ϩ sion by CD4 T cells, whatever the treatment of DCs (data not shown). The expression of various cytokine genes was quantified by ϩ shown). CD4 T cells produced IL-17A starting from day 7, with real-time PCR after stimulation of MoDCs obtained from a total of 26 35% of these cells secreting both IL-17A and IFN-␥. In cocultures healthy donors (Fig. 2A). In these experiments, a clear synergy be- performed with low responding MoDCs, only IFN-␥ was detected, tween LPS and R848 was observed in enhancing concomitantly the as early as day 3 (Fig. 1B, right panel). All results obtained by expression of IL-12p35, IL-12p40, IL-23p19, IL-6, IL-1␤, IL-10, and intracellular cytokine staining were confirmed when measuring se- ⌻⌵F-␣ genes in all donors, even if the magnitude of the up-regulation creted cytokines in culture supernatants (data not shown). varied dramatically between donors (Fig. 2A). A lower but substantial

ϩ up-regulation of these genes was still observed following stimulation The sequential polarization of CD4 T cell responses is due to with a single TLR agonist. MoDCs from eight high responding do- the synergistic up-regulation of cytokine production by MoDCs nors, i.e., confirmed to elicit a strong cytokine production by T cells, following stimulation via TLR4 and TLR7/8 exhibited a major increase in both IL-12p35 (Ͼ2115-fold), IL-12p40 To determine the cause of the differential CD4ϩ T cell polarization (Ͼ4286-fold), IL-23p19 (Ͼ1108-fold), IL-6 (Ͼ587-fold), IL-1␤ depending on MoDC donors, we further tested the effect of a single or (Ͼ37-fold), IL-10 (Ͼ13-fold), and ⌻⌵F-␣ (Ͼ16-fold) gene expres- combined TLR4 or TLR7/8 stimulation on MoDCs, evaluating sion following stimulation via TLR4 and TLR7/8 receptors (Fig. 2A, changes in surface phenotype, gene expression, and cytokine produc- open triangles). In MoDCs from low responders, the up-regulation of tion as read-outs. Whether used alone or in combination, both LPS cytokine genes following stimulation was usually significantly lower (TLR4 ligand) and R848 (TLR7/8 ligand) molecules induced a com- (Fig. 2A, black dots). Such differences between high and low respond- parable maturation of MoDCs, with a clear up-regulation of CD40, ers were confirmed following direct measurement of cytokine levels CD83, CD86, and HLA-DR surface molecules within 24 h (data not in culture supernatants from DCs (Fig. 2B). The Journal of Immunology 3377

FIGURE 6. R848 up-regulates IL-10, IL-12p35, IL-12p40, IL-23p19, and TLR7 gene expression in pDCs. Puriﬁed pDCs, magnetically sorted from human PBMCs, were stimulated for 24 h with R848 (1 ␮g/ml). A, IL-10, IL-12, IL-23, and TLR7 gene expression was analyzed by real-time PCR and normalized to the amount of RNA18S mRNA, with values for negative controls arbitrarily set to 1. Downloaded from B, Cytokine production at the protein level was measured by CBA for IL-10 and IL-12p70 and by ELISA for IL-23. Results obtained from 14 healthy donors are shown. MoDCs and pDCs isolated from healthy donors were stimulated for

24 h with either LPS plus R848 (1 ␮g/ml http://www.jimmunol.org/ each) or R848 (1 ␮g/ml), respectively. C, IL-12p35, IL-12p40, IL-23p19, and IL-10 gene expression were evaluated by real-time PCR. Levels of cytokine gene expression induced in the two DC subsets are shown as a correlation. by guest on September 23, 2021

We subsequently related cytokine gene expression by LPS plus black dots). Similarly, high responders exhibited higher baseline R848-treated MoDCs with cytokine induction in naive CD4ϩ T levels in TLR4 and TLR8 gene expression when compared with cells. Specifically, we noticed four clear correlations (i.e., between low responders (Fig. 4B), and the latter correlated with the level of IL-10 in MoDCs/IL-10 in T cells at day 1, IL-12p35 and IL-12p40 induction of IL-10, IL-12A, IL-12B, and IL-1␤ genes following in MoDCs/IFN-␥ in T cells at day 3, and IL-23 in MoDCs/IL-17 stimulation with either LPS or R848 (data not shown). in T cells at day 6) (Fig. 3A). These experiments also suggested Altogether, these results illustrated that a combined TLR4 and differences in terms of cytokine gene expression by LPS plus TLR7/8 stimulation of MoDCs triggers a major up-regulation of R848-treated MoDCs from low vs high responders (Fig. 3A, black both IL-12, IL-23, IL-6, IL-1␤, IL-10, and TNF-␣ production as dots and open triangles, respectively). well as an increased TLR7 and TLR8 expression in approximately To highlight the role of IL-10, IL-12, and IL-23 produced by one of three healthy donors tested. Polymorphisms observed in LPS plus R848-treated MoDCs in the sequential differentiation of those responses are probably related to a variation among donors naive CD4ϩ T cells, various blocking Abs were added to the co- in the capacity of TLR4 engagement to up-regulate TLR7 and cultures. As shown in Fig. 3B, the anti-IL-10 Ab up-regulated both TLR8 expression. IL-17A and IFN-␥ gene expression by T cells, the anti-IL-12 Ab decreased IFN-␥ gene expression whereas the anti-IL-23 Ab Role of TLR7 and TLR8 in the induction of IL-10, IL-12, and down-regulated IL-17A gene expression. IL-23 production by human DCs To inquire about possible mechanisms, we further analyzed To investigate the respective contribution of TLR7 and TLR8 in TLR4, 7, and 8 gene expression by MoDCs after LPS, R848, or the up-regulation of cytokines aforementioned, blocking experi- LPS plus R848 stimulation. Engagement of TLR4 or TLR7/8, ments with siRNAs specific for either TLR7 or TLR8 were per- alone or in combination, up-regulated both TLR7 and TLR8 genes. formed using high responding MoDCs treated with LPS plus Interestingly, TLR up-regulation after DC stimulation was consis- R848. The specificity of RNA silencing was confirmed using an tently higher in high vs low responders (Fig. 4A, open triangles vs irrelevant siRNA as a control (Fig. 5A). Silencing of either TLR7 3378 TLR7/8-STIMULATED DCs INDUCE COMPLEX T CELL DIFFERENTIATION

12p40 (Ͼ15-fold), IL-23 (Ͼ47-fold), and TLR7 (Ͼ12-fold) genes A (Fig. 6A, open triangles). Noteworthy, levels of cytokine mRNAs in TLR7 stimulated-pDCs were much lower when compared with MoDCs stimulated with TLR4 plus TLR7/8 ligands. We also compared patterns of cytokine gene expression observed in MoDCs stimulated by LPSϩR848 and pDCs incubated with R848 in four healthy donors. A clear correlation was observed for all donors, suggesting that the status of high/low responsiveness for a given donor extends to both MoDC and pDC subsets (Fig. 6C).

R848-treated pDCs trigger a differentiation of naive CD4ϩ T cells toward IFN-␥, IL-10, and IL-17 secreting cells To assess the impact of R848-treated pDCs on Th polarization, pDCs were stimulated with medium or R848 for 24 h, then washed and cocultured for up to 6 days with naive allogeneic CD4ϩ T cells. In cocultures performed with R848-treated pDCs from high responders (50% of donors), a strong induction of IFN-␥ gene

expression was detected at day 1, whereas IL-10 and IL-17A genes Downloaded from were only signiﬁcantly up-regulated after 6 days of coculture B (Fig. 7A, upper panel). Secretion of IL-10, IFN-␥, and IL-17A cytokines was conﬁrmed in 6-day culture supernatants (Fig. 7B, upper panel). As previously observed for MoDCs, low responding pDCs treated with R848 drove the differentiation of naive ϩ

CD4 T cells mostly toward IFN-␥-producing cells, with only http://www.jimmunol.org/ small amounts of IL-10 and IL-17A detected (Fig. 7, A and B, lower panels). In these experiments, neither gene expression nor cytokine production was detected in control conditions lacking T lymphocytes (data not shown).

Discussion Following recognition of viral or microbial pathogen associated molecular patterns, TLRs expressed by DCs critically influence the by guest on September 23, 2021 polarization of Ag-specific CD4ϩ T cell responses toward either FIGURE 7. R848-treated pDCs trigger IFN-␥, IL-10, and IL-17A pro- the Th1, Th2, Th17, or Treg lineages (3, 4). In this context, se- duction by naive CD4ϩ T cells. pDCs treated with R848 for 24 h were ϩ lected TLR ligands can be used alone or in combination, as po- washed and cultured for up to 6 days with naive allogeneic CD4 T cells. tential vaccine adjuvants to elicit the most appropriate immune Levels of cytokine gene expression were analyzed by real-time PCR. A, response in humans. Results obtained from six distinct donors are shown. Cytokine production was measured in corresponding supernatants by CBA for IFN-␥ and IL-10, In the present study, we observed, in agreement with a previous and ELISA for IL-17A. B, Allogeneic naive CD4ϩ T cells were cultured report (24), that a combination of TLR4 and TLR7/8 ligands acts with pDCs producing high or low (i.e., high or low responders) quantities in synergy to induce the production of multiple cytokines in of cytokines in response to R848. Data shown are the mean of six inde- MoDCs. The latter include IL-10, IL-12, and IL-23, known to ϩ p Ͻ 0.05. subsequently regulate distinct functional programs in naive CD4 ,ء .pendent experiments Ϯ SEM T cells associated with Treg (10), Th1 (13, 14), or Th17 differentiation (19), respectively. Levels of cytokine up-regulation in or TLR8 resulted in a decreased production of IL-10 and IL-12p70, MoDCs following joint stimulation via TLR4/TLR7/TLR8 dif- whereas it had no significant effect on IL-23 production (Fig. 5B). fered dramatically between healthy donors, even if some level of Dual silencing of TLR7 and 8 induced a strong down-regulation of synergy was always observed. One of three donors had MoDCs IL-10, IL-12p70, and IL-23, suggesting an additive and possibly exhibiting a strong up-regulation of cytokine genes. Such high synergistic role of these two TLRs in controlling the production of responding DCs were capable, when cultured with naive CD4ϩ T these cytokines (Fig. 5B). Although not shown, silencing of TLR7 cells, to induce a sequential production of IL-10, IFN-␥, and, even- and/or 8 had no impact on patterns of cytokines produced by tually, IL-17A, with peaks after 3, 5, and 7 days, respectively. MoDCs stimulated with LPS. pDCs stimulated through TLR7 alone also produced IL-10, IL-12, To extend these results, we assessed the impact of TLR7 stim- and IL-23. Interestingly, high responding pDCs elicited a distinct ulation on purified pDCs, which constitutively express high levels sequence of CD4ϩ T cell polarization when cultured with naive of this receptor, in the absence of TLR4 and TLR8. Stimulation CD4ϩ T cells, compared with LPS plus R848-treated MoDCs, with R848 alone was sufficient to enhance IL-10, IL-12p35, IL- with an initial IFN-␥-production followed by IL-17A and IL-10 12p40, IL-23, and IL-10 gene expression by pDCs (Fig. 6A), as secretion after 3 and 6 days, respectively. The reason for such a well as the production of corresponding cytokines (Fig. 6B). Based difference in the kinetics of cytokine secretion is presently unclear, on the observed variability in the responses among donors, it was but could be linked with differences in the regulation of IL-10, possible to distinguish high and low responders (Fig. 6, open triangles IL-12, and IL-23 gene expression in those two DC subpopulations. and black dots, respectively). High responding pDCs exhibited a par- Nonetheless, for a given donor the status of high/low responsive- allel up-regulation of IL-10 (Ͼ19.5-fold), IL-12p35 (Ͼ10-fold), IL- ness was found to extend to both MoDC and pDC subsets. The Journal of Immunology 3379

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