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• W2135327944 abstract "Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses. Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses. Dendritic cells (DCs) are key initiators of innate and adaptive immunity (for review, see Banchereau et al., 2000Banchereau J. Briere F. Caux C. Davoust J. Lebecque S. Liu Y.J. Pulendran B. Palucka K. Immunobiology of dendritic cells.Annu. Rev. Immunol. 2000; 18: 767-811Crossref PubMed Scopus (5458) Google Scholar, Lanzavecchia and Sallusto, 2001Lanzavecchia A. Sallusto F. The instructive role of dendritic cells on T cell responses: lineages, plasticity and kinetics.Curr. Opin. Immunol. 2001; 13: 291-298Crossref PubMed Scopus (322) Google Scholar, Mellman and Steinman, 2001Mellman I. Steinman R.M. Dendritic cells: specialized and regulated antigen processing machines.Cell. 2001; 106: 255-258Abstract Full Text Full Text PDF PubMed Scopus (1765) Google Scholar). They are potent antigen-processing and -presenting cells with the unique ability to stimulate primary immune responses in addition to boosting secondary immune responses. Immature DC precursors exit the bone marrow and circulate via the bloodstream to reach their target tissues, taking up residence at sites of potential pathogen entry in a physiological stage that is specialized for antigen capture (Steinman, 1991Steinman R.M. The dendritic cell system and its role in immunogenicity.Annu. Rev. Immunol. 1991; 9: 271-296Crossref PubMed Scopus (4212) Google Scholar). After uptake of antigen by phagocytosis, via macropinocytosis or via receptor-mediated endocytosis (Albert et al., 1998Albert M.L. Pearce S.F. Francisco L.M. Sauter B. Roy P. Silverstein R.L. Bhardwaj N. Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes.J. Exp. Med. 1998; 188: 1359-1368Crossref PubMed Scopus (1042) Google Scholar, Sallusto et al., 1995Sallusto F. Cella M. Danieli C. Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.J. Exp. Med. 1995; 182: 389-400Crossref PubMed Scopus (2130) Google Scholar), the antigen is processed and peptides thereof are presented on the cell surface associated with the MHC class I or II molecules. After receiving maturation-inducing signals, either directly from pathogens or via inflammatory stimuli, DCs change the expression pattern of chemokine receptors, allowing them to leave the peripheral tissue and to migrate to draining lymphoid organs (Rescigno et al., 1999Rescigno M. Granucci F. Citterio S. Foti M. Ricciardi-Castagnoli P. Coordinated events during bacteria-induced DC maturation.Immunol. Today. 1999; 20: 200-203Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Upregulation of CCR7 during maturation renders DCs sensitive to two chemoattractants, ELC/CCL19 and SLC/CCL21, which direct their migration to T cell regions of lymphoid organs (Cyster, 1999Cyster J.G. Chemokines and cell migration in secondary lymphoid organs.Science. 1999; 286: 2098-2102Crossref PubMed Scopus (843) Google Scholar, Sallusto and Lanzavecchia, 2000Sallusto F. Lanzavecchia A. Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression.Immunol. Rev. 2000; 177: 134-140Crossref PubMed Scopus (433) Google Scholar). Within these regions, DCs can induce both activation and proliferation of specific CTLs and Th cells via presentation of immunogenic peptides in association with MHC class I and II molecules, respectively. In addition to protection against pathogens, DCs also appear to be central to the regulation, maturation, and maintenance of cellular immune responses against cancer (Gunzer et al., 2001Gunzer M. Janich S. Varga G. Grabbe S. Dendritic cells and tumor immunity.Semin. Immunol. 2001; 13: 291-302Crossref PubMed Scopus (62) Google Scholar, Timmerman and Levy, 1999Timmerman J.M. Levy R. Dendritic cell vaccines for cancer immunotherapy.Annu. Rev. Med. 1999; 50: 507-529Crossref PubMed Scopus (422) Google Scholar). Recently, it has emerged that DCs not only induce T cell responses but are equally important for the maintenance of peripheral T cell tolerance because resting DCs induce T cell tolerance rather than immunity (Bonifaz et al., 2002Bonifaz L. Bonnyay D. Mahnke K. Rivera M. Nussenzweig M.C. Steinman R.M. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.J. Exp. Med. 2002; 196: 1627-1638Crossref PubMed Scopus (1048) Google Scholar, Probst et al., 2003Probst H.C. Lagnel J. Kollias G. van den Broek M. Inducible transgenic mice reveal resting dendritic cells as potent inducers of CD8+ T cell tolerance.Immunity. 2003; 18: 713-720Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Thus, regulation of DC activation is critical for the establishment of protective T cell responses as well as for the maintenance of T cell tolerance (for review, see (Bachmann and Kopf, 2002Bachmann M.F. Kopf M. Balancing protective immunity and immunopathology.Curr. Opin. Immunol. 2002; 14: 413-419Crossref PubMed Scopus (47) Google Scholar)). Pathogen-mediated DC activation is usually transduced via the Toll-like receptor (TLR) family (Akira, 2001Akira S. Toll-like receptors and innate immunity.Adv. Immunol. 2001; 78: 1-56Crossref PubMed Scopus (280) Google Scholar, Beutler, 2002Beutler B. Toll-like receptors: how they work and what they do.Curr. Opin. Hematol. 2002; 9: 2-10Crossref PubMed Scopus (126) Google Scholar, Medzhitov and Janeway, 2000Medzhitov R. Janeway Jr., C. The Toll receptor family and microbial recognition.Trends Microbiol. 2000; 8: 452-456Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). TLR recognize invariable patterns associated with pathogens like peptidoglycans (TLR2) (Takeuchi et al., 1999Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components.Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2720) Google Scholar), double-stranded RNA (TLR3) (Alexopoulou et al., 2001Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4698) Google Scholar), LPS (TLR4) (Hoshino et al., 1999Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.J. Immunol. 1999; 162: 3749-3752PubMed Google Scholar, Poltorak et al., 1998Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. et al.Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6242) Google Scholar), flagellin (TLR5) (Hayashi et al., 2001Hayashi F. Smith K.D. Ozinsky A. Hawn T.R. Yi E.C. Goodlett D.R. Eng J.K. Akira S. Underhill D.M. Aderem A. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5.Nature. 2001; 410: 1099-1103Crossref PubMed Scopus (2685) Google Scholar), and bacterial RNA (TLR 7/8) (Diebold et al., 2004Diebold S.S. Kaisho T. Hemmi H. Akira S. Reis E.S.C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science. 2004; 303: 1529-1531Crossref PubMed Scopus (2597) Google Scholar, Heil et al., 2004Heil F. Hemmi H. Hochrein H. Ampenberger F. Kirschning C. Akira S. Lipford G. Wagner H. Bauer S. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.Science. 2004; 303: 1526-1529Crossref PubMed Scopus (2863) Google Scholar) or DNA (TLR9) (Hemmi et al., 2000Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. A Toll-like receptor recognizes bacterial DNA.Nature. 2000; 408: 740-745Crossref PubMed Scopus (5175) Google Scholar, Schnare et al., 2000Schnare M. Holt A.C. Takeda K. Akira S. Medzhitov R. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88.Curr. Biol. 2000; 10: 1139-1142Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Activation of dendritic cells is also induced by proinflammatory cytokines and in particular by members of the TNF superfamily, including TNF-α, CD40L, and Trance/RANKL (Bachmann et al., 1999Bachmann M.F. Wong B.R. Josien R. Steinman R.M. Oxenius A. Choi Y. TRANCE, a tumor necrosis factor family member critical for CD40 ligand-independent T helper cell activation.J. Exp. Med. 1999; 189: 1025-1031Crossref PubMed Scopus (233) Google Scholar, Cella et al., 1996Cella M. Scheidegger D. Palmer-Lehmann K. Lane P. Lanzavecchia A. Alber G. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation.J. Exp. Med. 1996; 184: 747-752Crossref PubMed Scopus (1760) Google Scholar, Koch et al., 1996Koch F. Stanzl U. Jennewein P. Janke K. Heufler C. Kampgen E. Romani N. Schuler G. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.J. Exp. Med. 1996; 184: 741-746Crossref PubMed Scopus (844) Google Scholar, Wong et al., 1999Wong B.R. Josien R. Choi Y. TRANCE is a TNF family member that regulates dendritic cell and osteoclast function.J. Leukoc. Biol. 1999; 65: 715-724Crossref PubMed Scopus (196) Google Scholar). Recently, chemokine receptors have also been shown to be directly involved in induction of DC maturation. Specifically, activation through CCR5 by T. gondii-derived cyclophilin led to production of high levels of IL-12 in DCs (Aliberti et al., 2000Aliberti J. Reis e Sousa C. Schito M. Hieny S. Wells T. Huffnagle G.B. Sher A. CCR5 provides a signal for microbial induced production of IL-12 by CD8 alpha+ dendritic cells.Nat. Immunol. 2000; 1: 83-87Crossref PubMed Scopus (298) Google Scholar, Aliberti et al., 2003Aliberti J. Valenzuela J.G. Carruthers V.B. Hieny S. Anderson J. Charest H. Reis e Sousa C. Fairlamb A. Ribeiro J.M. Sher A. Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells.Nat. Immunol. 2003; 4: 485-490Crossref PubMed Scopus (194) Google Scholar). The importance of DC maturation for the generation of protective T cell responses is also highlighted by the fact that several viruses interfere with activation of DCs. Poxviruses inhibit DC maturation by lowering the expression of CD86, leading to reduced T cell costimulation (Engelmayer et al., 1999Engelmayer J. Larsson M. Subklewe M. Chahroudi A. Cox W.I. Steinman R.M. Bhardwaj N. Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion.J. Immunol. 1999; 163: 6762-6768PubMed Google Scholar). Along a similar line, herpes simplex virus inhibits maturation of DCs (Salio et al., 1999Salio M. Cella M. Suter M. Lanzavecchia A. Inhibition of dendritic cell maturation by herpes simplex virus.Eur. J. Immunol. 1999; 29: 3245-3253Crossref PubMed Scopus (324) Google Scholar), and measles virus induces an aberrant maturation program resulting in inhibition rather than activation of T cells (Schneider-Schaulies et al., 2003Schneider-Schaulies S. Klagge I.M. ter Meulen V. Dendritic cells and measles virus infection.Curr. Top. Microbiol. Immunol. 2003; 276: 77-101PubMed Google Scholar). To identify novel proteins capable of regulating T cell responses through the activation of DCs, we performed an alphavirus based expression cloning campaign. We used Sindbis virus as a carrier of a normalized cDNA library from activated splenocytes and generated a protein library by infection of baby hamster kidney (BHK) cells (Koller et al., 2001Koller D. Ruedl C. Loetscher M. Vlach J. Oehen S. Oertle K. Schirinzi M. Deneuve E. Moser R. Kopf M. et al.A high-throughput alphavirus-based expression cloning system for mammalian cells.Nat. Biotechnol. 2001; 19: 851-855Crossref PubMed Scopus (25) Google Scholar). Sindbis virus belongs to the alphavirus genus and contains a single-stranded RNA genome with positive polarity. Using a bipartite system, where one RNA molecule encodes the structural proteins and the other encodes the protein of interest plus the viral replicase, it is possible to generate replication competent viruses encoding genes or even libraries of interest (Koller et al., 2001Koller D. Ruedl C. Loetscher M. Vlach J. Oehen S. Oertle K. Schirinzi M. Deneuve E. Moser R. Kopf M. et al.A high-throughput alphavirus-based expression cloning system for mammalian cells.Nat. Biotechnol. 2001; 19: 851-855Crossref PubMed Scopus (25) Google Scholar). We report here the identification of mouse CCL19 and CCL21 as potent maturation factors for DCs and indirect regulators of Th cell differentiation. Our findings indicate that induction of DC maturation is an important property of CCL19/CCL21 and suggest that chemokines may not only organize the migration of DCs but also directly regulate their ability to prime T cell responses. Because DCs are crucial for the induction of immune responses, we set out to identify novel DC maturation factors using an alphaviral screening technology. In order to generate a Sindbis virus-based cDNA library, RNA was extracted from spleens of LCMV-infected BALB/c mice, transcribed into cDNA and normalized as described in the Experimental Procedures. The cDNA library was transformed into a viral library by in vitro transcription and electroporation together with a viral helper construct (Koller et al., 2001Koller D. Ruedl C. Loetscher M. Vlach J. Oehen S. Oertle K. Schirinzi M. Deneuve E. Moser R. Kopf M. et al.A high-throughput alphavirus-based expression cloning system for mammalian cells.Nat. Biotechnol. 2001; 19: 851-855Crossref PubMed Scopus (25) Google Scholar). Following the strategy shown in Figure 1A , BHK cells were infected at a multiplicity of infection (MOI) of 0.1 and single infected cells were sorted on subconfluent BHKs in 96-well plates. This led to the generation of a protein library within 3 days, where each well corresponded to the expression of one cDNA-derived protein. The protein-containing supernatants were harvested, and remaining virus was inactivated by UV light. Spleen-derived DCs were pulsed with OVA peptide and treated with the supernatants overnight. The next day, OVA-specific CD4+ T cells were added, and proliferation was assessed 2.5 days later by 3H-thymidine incorporation. The average counts per plate were calculated and every supernatant that induced a count/min value two standard deviations above the average was retested by the same assay. From those wells that scored positive a second time, the virus-encoded cDNA was recovered by RT PCR, and the cDNA sequence was determined (Figure 1B). In total 10,000 genes derived from the library were tested for their ability to enhance T cell proliferation in the assay. Besides known inducers of DC maturation, like hsp70, the chemokine CCL19 was identified twice independently in this screening campaign. One of the plates in which CCL19 was identified is shown in Figure 1B. To confirm that CCL19 induces T cell proliferation in the DC-T cell coculture system, the CCL19 cDNA was cloned into the alphaviral vector, and the recombinant virus was generated (SR5-CCL19). BHK cells were infected, and the supernatant was retested in the proliferation assay. In these experiments, the proliferation-stimulating activity of CCL19 was confirmed (Figure 2A ). In order to assess whether CCL19 directly induces T cell proliferation, naïve T cells were incubated in the presence or absence of CCL19. No T cell proliferation was induced by CCL19 (data not shown), suggesting that the observed induction of T cell proliferation is mediated solely through enhancement of DC maturation. To test this further, spleen-derived DCs were treated overnight with the supernatants and analyzed for surface expression of CD40 and CD86 by flow cytometry. A significantly higher proportion of mature DCs were observed in cultures treated with supernatants from SR5-CCL19-infected BHK cells compared to DC cultures treated with supernatants from control virus-infected BHK cells (Figures 2B and 2C). As a positive control, we included Sindbis virus-expressing TNF-α, which enhanced T cell proliferation (Figure 2A) and induced DC maturation in a manner comparable to CCL19 (Figures 2B and 2C). To test whether the effects found where specific for CCL19 and not due to the use of a viral expression system, an N-terminal FLAG-tagged CCL19 protein was produced in HEK 293 cells and purified over an anti-FLAG column (Figure 3A ). This purified protein was used for all further experiments. First, the proliferation promoting quality in the DC-T cell coculture system was retested. Bone marrow-derived DCs were taken as APCs, and proliferation was tested at three different OVA peptide concentrations. DCs were pulsed with the peptide for 1 hr and then treated with the different stimuli for 4 hr. OVA-specific T cells were subsequently added and proliferation was measured 3 days later (Figure 3B). In a similar experiment, T cell proliferation was assessed by CSFE staining. DCs were treated as above and incubated for 4 days with CSFE-labeled T cells. In both experiments, CCL19 enhanced T cell proliferation to the same extent as treatment with LPS plus anti-CD40 antibodies or TNF-α (Figure 3C). The maturation of DCs is associated with the loss of receptors involved in antigen uptake and the upregulation of costimulatory molecules that are important for productive interaction with T cells. Through the concomitant production of cytokines, DCs can further promote the induction of an immunological response following pathogen exposure. In order to analyze the maturation program triggered in DCs by CCL19, we examined the surface expression of CD40, CD80, CD86, MHC II, and, additionally, the levels of the proinflammatory cytokines interleukin-1 beta (IL-1β), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-α). In the first instance, DCs were treated overnight and the surface expression of CD40, CD80, CD86, and MHC II was assessed by flow cytometry (Figures 4A and 4B and Figure S1 in the Supplemental Data available with this article on line). To exclude that the observed effect was due to LPS contamination, the samples were treated with proteinase K and subsequent heat denaturation. CCL19 was found to induce upregulation of CD86 (Figure 4A) and CD40 (Figure 4B) in an LPS-independent fashion, shown by a loss of function following proteinase K treatment. Similar upregulation of CD86 and CD40 was found for CCL19 as for TNF-α and LPS treatment (Figures 4A and 4B). To further ensure that the DC maturation effect was mediated by CCL19, we tested its activity on DCs genetically deficient in the CCL19 receptor, CCR7. The CCL19-induced DC maturation was indeed mediated through binding to CCR7 because production of IL-12 (Figure 4C) and upregulation of CD86, CD80, and CD40 surface expression was not evident after stimulation of CCR7−/− DC by CCL19 (Figure 4D). Of note, the activity of CCL19 was Pertussis toxin sensitive and also independent of MyD88 (data not shown). We next performed titration experiments and found that CCL19 induced DC maturation at concentrations of 0.1 μg/ml and higher (Figures 4E and 4F), roughly corresponding to chemokine concentrations used to induce cellular migration (Figure 4G) (see also Kwan and Killeen, 2004Kwan J. Killeen N. CCR7 directs the migration of thymocytes into the thymic medulla.J. Immunol. 2004; 172: 3999-4007Crossref PubMed Scopus (145) Google Scholar, Moutaftsi et al., 2004Moutaftsi M. Brennan P. Spector S.A. Tabi Z. Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells.J. Virol. 2004; 78: 3046-3054Crossref PubMed Scopus (53) Google Scholar). Two commercially available CCL19 batches produced in a prokaryotic expression system were also tested and found to be less active. This limited activity correlated with a roughly 100-fold reduced ability of the commercial CCL19 to induce migration of DCs (data not shown). Thus, in our experimental system, commercial bacterially expressed CCL19 appears to have an overall limited potency. To test whether CCL19 induced secretion of relevant inflammatory cytokines, supernatants of the stimulated DCs were analyzed for the presence of IL-1β and IL-12 (p40) by ELISA. CCL19 induced strong production of both cytokines (Figure 5A ). Induction of IL-12 production was similar to LPS-treated DCs, whereas IL-1β production was even higher than that seen after LPS stimulation. In comparison to TNF-α, CCL19 induced clearly higher levels of both IL-12 and IL-1β. In additional experiments, we compared induction of inflammatory cytokines by CCL19 with combined stimulation of DCs with LPS and anti-CD40 antibodies. This stimulus has recently been shown to be sufficient for induction of autoimmunity by using peptide-pulsed DCs (Eriksson et al., 2003Eriksson U. Ricci R. Hunziker L. Kurrer M.O. Oudit G.Y. Watts T.H. Sonderegger I. Bachmaier K. Kopf M. Penninger J.M. Dendritic cell-induced autoimmune heart failure requires cooperation between adaptive and innate immunity.Nat. Med. 2003; 9: 1484-1490Crossref PubMed Scopus (318) Google Scholar). Cytokine production was assessed by intracellular cytokine staining (Figure 5B). Surprisingly, CCL19 induced similar levels of IL-1β, TNF-α, and IL-12 as compared to stimulation with LPS plus anti-CD40 antibodies, confirming that CCL19 is indeed a strong maturation trigger for DCs (Figure 5B). To test whether the second ligand of CCR7, CCL21, also induces maturation of DCs, we expressed and purified a FLAG-tagged version of CCL21 as described for CCL19. Bone marrow-derived dendritic cells were then stimulated with titrated amounts of FLAG-CCL21 and analyzed for the expression of CD86 and CD40. As observed for CCL19, CCL21 was able to strongly stimulate DC maturation at concentrations above 0.1 μg/ml (Figure S2). We sought to determine whether CCL19 stimulation of DCs would preferentially facilitate the induction of either Th1 or Th2 responses. Accordingly, we employed the use of a DC-transgenic T cell coculture system where T helper cell differentiation is controlled by peptide concentration (Marsland et al., 2004Marsland B.J. Soos T.J. Spath G. Littman D.R. Kopf M. Protein kinase C theta is critical for the development of in vivo T helper (Th)2 cell but not Th1 cell responses.J. Exp. Med. 2004; 200: 181-189Crossref PubMed Scopus (186) Google Scholar, Ruedl et al., 2000aRuedl C. Bachmann M.F. Kopf M. The antigen dose determines T helper subset development by regulation of CD40 ligand.Eur. J. Immunol. 2000; 30: 2056-2064Crossref PubMed Scopus (112) Google Scholar). In this coculture system, high concentrations of peptide result in Th1 differentiation and IFN-γ production, while low concentrations of peptide result in Th2 differentiation and IL-4 production. Dendritic cells were incubated for 4 hr in the presence or absence of CCL19, washed twice, and used to stimulate CD4+ TCR-transgenic T cells from SMARTA mice (Oxenius et al., 1998Oxenius A. Bachmann M.F. Zinkernagel R.M. Hengartner H. Virus-specific MHC-class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection.Eur. J. Immunol. 1998; 28: 390-400Crossref PubMed Scopus (284) Google Scholar) specific for peptide GP13 derived from LCMV. To ascertain whether CCL19 was capable of directly activating T cells, naïve T cells were also incubated for 4 hr in the presence or absence of CCL19 before use in the coculture. A peptide concentration of 100 nM was used in these cultures because this induces differentiation of both Th1 and Th2 cells, allowing any additional polarizing effects mediated by CCL19 to be identified. After 3 days of culture, production of IL-4 and IFN-γ by the transgenic CD4+ T cells was assessed by intracellular cytokine staining. As expected, untreated DCs pulsed with 100 nM GP13 induced both Th1 and Th2 cells, as shown by the presence of both IL-4- and IFN-γ-producing cells (Figure 6, left panel). In marked contrast, DCs activated with CCL19 preferentially mediated Th1 development, increasing the proportion of IFN-γ-producing cells and decreasing the proportion of IL-4-producing cells (Figure 6, middle panel). Preincubating T cells with CCL19 did not influence T cell differentiation, indicating that the effect of CCL19 was limited to the activation of DCs (Figure 6, right panel). Taken together, these data show that CCL19 selectively mediates induction of Th1 responses. In addition to the previously described chemoattractive properties of CCL19, we have shown that it can directly activate DCs and lead to the production of proinflammatory cytokines and upregulation of costimulatory molecules. However, the constitutive production of CCL19 within lymphoid tissues would not be expected to result in the activation of steady-state DCs because autoimmunity would otherwise result. To examine this directly, we compared CCR7 expression on semimature/steady-state DCs in the lymph node to that of DCs that had been activated in the periphery by a TLR ligand and had migrated to the lymph node to present exogenous antigen. We administered FITC-conjugated OVA protein (FITC-OVA) and LPS via the intranasal route, and 24 hr later, we removed the draining lymph node and determined which cell populations contained FITC-OVA. The only cell population positive for FITC-OVA were CD11chigh MHC IIhigh DCs (Figure 7A ), consistent with active transport of the antigen rather than passive drainage where additional cell populations would be expected to take-up OVA. Also present in the draining lymph node were semimature/steady-state DCs that expressed CD11chigh/int and MHC IIint but were negative for FITC-OVA (Figure 7A). We assessed the level of CCR7 surface expression on these two DC populations and found that the LPS-activated, OVA-presenting CD11chigh MHC IIhigh DCs expressed higher levels of CCR7 than the semimature/steady-state DCs (Figure 7A). To determine whether CCR7high TLR ligand-activated OVA-presenting DCs exhibited a comparable response to CCL19 as the CCR7low DCs, we next sorted the respective DC populations by FACS then activated them for 6 hr with either CCL19 or LPS, and we then assessed the production of IL-12, IL-10, or the surface expression of CD40. The TLR ligand-activated OVA-presenting DCs produced moderate levels of IL-12 without further stimulation ex vivo, reflective of their activated in vivo phenotype and possibly an increased sensitivity to activation during the isolation and sorting procedure (Figure 7B). Comparatively, the proportion of CCR7low DCs that produced IL-12 without further stimulation ex vivo was 6-fold lower (Figure 7B). Upon CCL19 or LPS activation, the TLR ligand-activated OVA-presenting DCs produced high levels of IL-12 and upregulated CD40 expression, while the CCR7low DCs exhibited no response to CC" @default.
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• W2135327944 date "2005-04-01" @default.
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• W2135327944 title "CCL19 and CCL21 Induce a Potent Proinflammatory Differentiation Program in Licensed Dendritic Cells" @default.
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• W2135327944 doi "https://doi.org/10.1016/j.immuni.2005.02.010" @default.
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