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 References This article cites 28 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/182/6/3372.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 consti- tutively 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 Franc¸ais 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 produc- tion by MoDCs. Human MoDCs ob- tained from 26 healthy donors were cul- tured 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).