CC- Ligand 16 Induces a Novel Maturation Program in Human Immature -Derived Dendritic Cells

This information is current as Paola Cappello, Tiziana Fraone, Laura Barberis, Carlotta of October 1, 2021. Costa, Emilio Hirsch, Angela R. Elia, Cristiana Caorsi, Tiziana Musso, Francesco Novelli and Mirella Giovarelli J Immunol 2006; 177:6143-6151; ; doi: 10.4049/jimmunol.177.9.6143 http://www.jimmunol.org/content/177/9/6143 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CC-Chemokine Ligand 16 Induces a Novel Maturation Program in Human Immature Monocyte-Derived Dendritic Cells1

Paola Cappello,*† Tiziana Fraone,* Laura Barberis,‡ Carlotta Costa,‡ Emilio Hirsch,‡ Angela R. Elia,*¶ Cristiana Caorsi,*¶ Tiziana Musso,§ Francesco Novelli,*¶ and Mirella Giovarelli2*¶

Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host’s defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through

differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are Downloaded from characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the

migration of MoDCs, but also directly regulate their ability to prime T cell responses. The Journal of Immunology, 2006, 177: http://www.jimmunol.org/ 6143–6151.

endritic cells (DCs)3 are the most important link be- Furthermore, DCs are affected by endogenous mediators released tween the innate and the acquired immune response and by infected, damaged, or inflamed tissues, such as proinflamma- D are involved in the initiation of both types of immunity tory , prostaglandin, or catabolites from dying cells (5, (1). Immature DC (iDC) precursors exit from the bone marrow, 6). Chemokine receptors have also been shown to be directly in- circulate via the bloodstream to reach their target tissues, and take volved in induction of DC maturation. Specifically, activation up residence at sites of potential pathogen entry in a physiological through CCR5 by Toxoplasma gondii-derived cyclophilin leads to by guest on October 1, 2021 stage that is specialized for Ag capture (1). Tissue injury and in- production of high levels of IL-12 in DCs (7, 8). Little is known, flammation cause dramatic changes throughout the microenviron- on the other hand, about chemokines and DC maturation. We fo- ment, including the release of inflammatory mediators such as cy- cused our attention on CCL16/-expressed chemokine’s effects tokines and chemokines that shape protective immune responses because we had demonstrated its potent antitumor activity associ- and culminate in pathogen elimination. Many stimuli are involved ated with recruitment of leukocytes (9). CCL16 is chemotactic for in DC maturation (2). DCs receive information directly from monocytes, , eosinophils, and DCs (for a review, see pathogens via “pattern-recognition receptors” such as the TLRs (3) Zlotnik et al. (10)). It binds CCR1, CCR2, CCR5, and CCR8 (11, and from cytokines released by T cells during their differentiation 12) and has been used to treat established murine tumors. CCL16 toward Th1 or Th2 in peripheral tissues or lymph nodes (LNs) (4). expressed by adenoviruses (13, 14) or as recombinant fusion pro- tein (15) induces accumulation of T cells and DCs at the tumor site *Center for Experimental Research and Medical Studies, San Giovanni Battista Hos- pital, Turin, Italy; †Department of Clinical and Biological Sciences, University of and in draining LNs. Turin, Orbassano, Italy; ‡Department of Genetics, Biology, and Biochemistry, §De- Our previous data have shown that CCL16 costimulates mac- ¶ partment of Public Health and Microbiology, and Department of Medicine and Ex- rophage cytotoxic and Ag-presenting functions in the mouse (16). perimental Oncology, University of Turin, Turin, Italy CCL16 released by lamina propria-infiltrating macrophages elicits Received for publication November 22, 2005. Accepted for publication August 14, 2006. massive recruitment of immune reactive cells in ulcerative colitis The costs of publication of this article were defrayed in part by the payment of page (17). These results led us to study the effects of CCL16 on human charges. This article must therefore be hereby marked advertisement in accordance DCs derived from monocytes (MoDCs) in the presence of GM- with 18 U.S.C. Section 1734 solely to indicate this fact. CSF and IL-4. We report here that human CCL16 is a potent mat- 1 This work was supported by the Italian Association for Cancer Research, the Italian Ministry for Education, the Universities and Research, Fondi incentivazione della uration factor for MoDCs through differential use of its four re- Ricerca di Base and Progetti di Ricerca di Interesse Nazionale, and Compagnia San ceptors and an indirect regulator of Th cell differentiation. We Paolo, Special Project Oncology. analyzed the expression of costimulatory molecules and chemo- 2 Address correspondence and reprint requests to Professor Mirella Giovarelli, De- kine receptors and confirmed their functional efficiency. We also partment of Experimental Medicine and Oncology, Corso Raffaello 30, 10125 Torino, Italy. E-mail address: [email protected] evaluated the ability of MoDCs induced to mature by CCL16 3 Abbreviations used in this paper: DC, dendritic cell; CCL16/mDC, MoDC induced (CCL16/mDCs) to induce alloactivation of T cells and take up Ag. to mature in the presence of CCL16; iDC, immature DC; LN, lymph node; mDC, DC Our findings suggest that chemokines may not only organize the induced to mature in the presence of TNF-␣ plus IL-1␤; MFI, mean fluorescence intensity; MoDC, monocyte-derived DC; PLC, phospholipase C; PTX, pertussis migration of DCs but also directly regulate their ability to prime T toxin. cell responses.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 6144 MoDC MATURED BY CCL16 CAN STILL TAKE UP Ag

Materials and Methods Mixed reaction CCL16 source T cells were purified on a nylon wool column (Robbins Scientific) and ϫ 5 The human recombinant CCL16 used in these experiments (PeproTech) is placed in 96-well plates at 1 10 cells/well with allogeneic iDCs and differentially mature DCs, after 24 h of stimulation with CCL16 or TNF-␣ a 11.2-kDa protein of 97-aa residues produced in Escherichia coli from a and IL-1␤, in increasing concentrations (150–10, 000). After 4 days, 1 ␮Ci DNA sequence encoding the mature human CCL16 protein sequence 3 (Q26-Q120). Its endotoxin level is Ͻ0.1 ng/␮g(1EU/␮g) as determined of tritiated [ H]TdR (Amersham Biosciences) was added to each well and incubation was prolonged for an additional 18 h. Cells were directly col- by the Limulus Amebocyte Lysate assay. lected with a CellHarvester (Packard Instrument) in UNIfilter plates (Pack- ard Instrument) and [3H]TdR uptake was quantitated (TopCount micro- Generation and maturation of MoDCs plates scintillation counter; Packard Instrument). All tests were performed Human PBMCs were isolated from venous blood of voluntary healthy in triplicate. donors by Ficoll-Paque density gradient centrifugation (Pharmacia Bio- Supernatants from T cells cultured with iDCs, CCL16/mDCs, or mDCs at ratio 20:1 T:DC were harvested after 48 h and analyzed by ELISA for tech). Monocytes were enriched with an isolation kit (Miltenyi Biotec). ␥ The resulting preparations were consistently Ͼ90% CD14ϩ as determined IFN- and IL-4 production (both R&D Systems) in accordance with the by FACSCalibur (BD Biosciences). To prepare iDCs, the enriched mono- manufacturer’s protocol. ϫ 6 cytes were incubated in 6-well culture plates (5 10 cells/3 ml/well) in detection AIM V medium (Invitrogen Life Technologies) supplemented with 100 ng/ml GM-CSF and 50 ng/ml IL-4 (both PeproTech) for 6 days. On days Production of cytokines and chemokines in the supernatants was evaluated 2 and 4, two-thirds of the culture medium were replaced by fresh medium with a commercial RayBio Human Cytokine Array V (Tebu-bio) in accor- containing GM-CSF and IL-4. iDCs were resuspended as 1 ϫ 106 cells/ml dance with the manufacturer’s protocol. The membranes were exposed to in AIM V supplemented or not with CCL3, CCL4, CCL14, and CCL16 (all x-ray films and signals were detected with a film developer (Kodak Hy- from PeproTech) at 1000 and 100 ng/ml, as indicated, or human TNF-␣ (50 perfilm; Amersham Biosciences). Significance was increased by using Downloaded from ng/ml) plus IL-1␤ (50 ng/ml) (both from PeproTech). Their endotoxin level pools of supernatants from single donors of iDCs, CCL16/mDCs and is Ͻ0.1 ng/␮g as determined by the Limulus Amebocyte Lysate assay. mDCs. The signal intensities were quantitated by densitometry with After 24 or 48 h, as indicated, cells were harvested and analyzed. UTHSCSA ImageTool, a free image processing and analysis program, and positive controls present on the membranes were used to normalize the Immunophenotypic analysis results for comparison. An increase or decrease of 30% was considered significantly different. After 24 h in the presence of medium only, CCL16 (at both 100 and 1000 Supernatants from iDCs, CCL16/mDCs, and mDCs from 12 donors ng/ml), or TNF-␣ and IL-1␤, cells were washed and resuspended in PBS were analyzed by ELISA for IL-10 and CXCL10 (both from R&D Sys- http://www.jimmunol.org/ (Sigma-Aldrich) supplemented with 0.2% BSA and 0.01% sodium azide, tems) secretion in accordance with the manufacturer’s protocol. and incubated with fluorochrome-conjugated mAbs and isotype-matched negative controls (DakoCytomation) after blocking nonspecific sites with Ag uptake assay rabbit IgG (Sigma-Aldrich) for 30 min at 4°C. The following mAbs were ␣ ␤ used: anti-CD14 (BD Biosciences); anti-CD1a, anti-CD80, anti-CD83, anti- After 24 h of stimulation with CCL16 or TNF- and IL-1 , iDCs and ϫ 6 CD86, anti-HLA-DP, -DQ, and -DR (Ancell); and anti-CCR5, anti-CCR6, differentially mature DCs were incubated at 1 10 cells/ml in AIM V for anti-CCR7, and anti-CXCR4 (R&D Systems). Samples were collected and 15 min at 4 or 37°C. FITC-labeled dextran (Mr 70,000; Sigma-Aldrich) was analyzed using a FACSCalibur CellQuest (BD Biosciences). Cells were added at the final concentration of 1 mg/ml, and the cells were incubated electronically gated according to their light-scatter properties to exclude for 15, 30, 60, and 120 min to allow capture of Ag. After thorough washing cell debris and contaminating lymphocytes. To determine which CCL16 with cold PBS, fluorescence was measured by FACSCalibur CellQuest receptor is involved in the CCL16-induced maturation of MoDCs, iDCs analysis to reveal dextran uptake. by guest on October 1, 2021 were pretreated with mAb to CCR1 (MBL International), CCR2 (R&D Systems), CCR5 (BD Biosciences), or CCR8 (R&D Systems), or an iso- Statistical analysis type control IgG (DakoCytomation) for 30 min at 4°C and, without wash- The significance of differences in counts per minute obtained from the ing, incubated in the presence or not of CCL16. [3H]TdR uptake assay, in migration in the presence of stimuli, and in cy- To determine the signaling pathway effective in the CCL16-induced tokine secretion was evaluated with an unpaired Student’s t test (GraphPad maturation of MoDCs, iDCs were pretreated with inhibitors, namely, per- Prism 3.1; GraphPad Software). tussis toxin (PTX) from Bordetella pertussis (100 ng/ml; Sigma-Aldrich), LY290042 (25 ␮M; Sigma-Aldrich), wortmannin from Penicillium funicu- losum (200 nM; Sigma-Aldrich), SB203580 (20 ␮M; Sigma-Aldrich), Results U73122 (1 ␮M; Sigma-Aldrich), or PD98058 (50 ␮M; Calbiochem) for 45 CCL16 induces migration of MoDCs by binding its four min at 37°C and, without washing, incubated in the presence or absence of receptors CCL16. After 24 h, cells were processed as indicated above. The percent inhibition was calculated as (100 Ϫ (mAb treatment ϩ CCL16/IgG ϩ CCL16 is chemotactic for human DCs (10), especially at 1000 CCL16) ϫ 100) or (100 Ϫ (inhibitor ϩ CCL16/0, 1% DMSO ϩ ng/ml. We therefore set out to determine which receptor is in- CCL16) ϫ 100). volved in this migration by pretreating (or not) iDCs with neutral- izing Abs to CCR1, CCR2, CCR5, and CCR8 and assessing their ability to migrate in response to CCL16 at 1000 ng/ml. In each of Boyden chambers (NeuroProbe) were filled with stimulus (CCL1, CCL2, three independent experiments we used the specific ligands for CCL4, CCL14, CCL16, CCL20, CXCL12, or CCL19; all from PeproTech) each CCR as the positive control. As shown in Fig. 1, CCL16 diluted in RPMI 1640 (BioWhittaker Europe) 1% FCS certified (Invitrogen significantly induced migration of iDCs, while addition of the neu- Life Technologies) at reported concentrations, and only medium as control, tralizing mAbs to CCR5 (100%) and CCR8 (73%) and (to a lesser and covered with polycarbonate filters (pore size 5 ␮m; Nucleopore). After 24 h of stimulation in the presence or absence of CCL16 or TNF-␣ and extent) CCR1 and CCR2 (56–63%) inhibited this migration. The IL-1␤,50␮l of MoDC suspension in RPMI 1640–1% FCS at a concen- presence of all four mAbs inhibited iDC migration less (94%) than tration of 1 ϫ 106 cells/ml was added to each chamber. After2hat37°C, anti-CCR5 alone. Addition of an unrelated mAb had no effect on cells that had migrated to the lower part of the filter were stained with this migration. CCL16, therefore, uses all its receptors to induce Giemsa-May-Gru¨nwald (Diff-Quik kit; Dade Behring) and counted under a microscope (objective immersion oil, ϫ100; Olympus). To determine iDC migration, probably with different affinity. which receptor is involved in the CCL16-induced migration of iDCs, they were pretreated with 10 ␮g/ml neutralizing mAb against CCR1, CCR2, CCL16 induces maturation of human MoDCs CCR5, and CCR8 or isotype control Ig for 30 min at 4°C and then resus- After only 24 h of stimulation with 100 and 1000 ng/ml chemokine pended in medium only. These cells were incubated in a Boyden chamber in the presence or absence of 1000 ng/ml CCL16 for2hat37°C, as concentrations, CCL16/mDCs displayed a higher expression of described above. CCL1, CCL2, CCL4, and CCL14 were used as positive CD80 and CD86 costimulatory molecules and MHC class II mol- controls. ecules compared with iDCs (Fig. 2). The maturation marker CD83 The Journal of Immunology 6145

higher concentration more efficiently induced their increased ex- pression as shown by the mean fluorescence intensity (MFI) and was therefore used in all the other experiments. Because DC maturation is associated with differential regulation of chemokine receptors (18), we determined whether CCL16 mod- ifies their expression pattern on MoDCs. CCL16/mDCs, in fact, up-regulated receptors CXCR4 and CCR7 but still maintained the expression of CCR5 (Fig. 2). Interestingly, the CCR5 receptor was found on iDCs generated in vitro (Fig. 2), whereas mDCs down-modulated CCR5 and pri- marily expressed CXCR4 and CCR7. CCL16/mDCs differ from mDCs in their persistence of CCR5 expression and ex novo CCR6 expression, which is usually found on CD34ϩ-derived DCs and Langerhans DCs and is important for the recruitment of iDCs to an injury site. FIGURE 1. CCL16 induces migration of MoDCs. iDCs pretreated or CCL3, CCL4, and CCL14 share the same putative receptors as not with neutralizing Abs against CCR1, CCR2, CCR5, CCR8, or all to- CCL16. When they were used as maturation stimuli, only CCL14, gether, or with an isotype control Ig for 30 min at 4°C were incubated in a CCR1 agonist, displayed a similar ability to induce CD83 and a Boyden chamber in the presence or not of CCL16 (1000 ng/ml) for 2 h CCR7 maturation markers (Table I). The influence of trace Ϯ Downloaded from at 37°C. The results are the mean SD of migrated cells/field from three amounts of endotoxin in the chemokine preparations can be ruled p Ͻ 0.05, values from mAbs ,ء .experiments with three independent donors out because all are claimed to contain Ͻ0.1 ng/␮g endotoxin. Even pretreated cells compared with untreated cells that migrated in the presence .p Ͻ 0.05, values from cells that migrated in the presence of so, only CCL16 and CCL14 induced maturation markers on iDCs ,ءء ;of CCL16 CCL16 compared with cells migrated in the absence of stimuli. Moreover, boiled CCL16 did not stimulate phenotypic changes in iDCs (data not shown). To find out which receptor is involved in the CCL16-induced http://www.jimmunol.org/ was expressed by a substantial percentage of CCL16/mDCs but maturation of human MoDCs, we pretreated iDCs with neutraliz- was undetectable in iDCs. As shown in Fig. 2, both CCL16 con- ing mAbs to CCR1, CCR2, CCR5, or CCR8 for 30 min at 4°C, centrations exerted similar effects, although for some markers, the followed by incubation in the presence or absence of CCL16 for by guest on October 1, 2021

FIGURE 2. CCL16 induces maturation of MoDCs. iDCs (first and fourth rows) were cultured for an addi- tional 24 h with CCL16 at 100 and 1000 ng/ml (CCL16/ mDCs, second and fifth rows, gray peak and thick solid line, respectively) or TNF-␣ and IL-1␤ (mDCs, third and sixth rows). CCL16/mDCs acquire a mature DC pheno- type (HLA-DRhigh, CD83ϩ, CD80high, CD86high, CXCR4ϩ, and CCR7ϩ) but maintain CCR5 and express ex novo CCR6. The thin solid line shows the isotype con- trol. One of five independent experiments with different donors is shown. A, HLA-II, CD83, CD80, CD86 and B, chemokine receptors surface expression. 6146 MoDC MATURED BY CCL16 CAN STILL TAKE UP Ag

Table I. CD83 and CCR7 expression by stimulated iDCs Table III. Signal pathways involved in the CCL16-induced maturation of human MoDCs Stimulia CD83 CCR7 Percentage of Inhibitiona of 8b 1 CCL3 7 5 Inhibitor CD83 CD86 CXCR4 CCR6 CCL4 7 24 CCL14 36 34 PTX 0 30 0 100 CCL16 54 57 LY294002 31 90 85 0 TNF-␣ plus IL-1␤ 80 65 Wortmannin 0 42 0 100 PD98059 0 17 0 0 a Chemokines were added to iDCs at 1000 ng/ml, TNF-␣ and IL-1␤ at 50 ng/ml, SB203580 53 51 100 84 and the cells were harvested and analyzed by flow cytometry after 24 h (see Materials U73122 34 66 85 84 and Methods). b Percentage of positive cells. One of three independent experiments with different a Inhibition up to 25% is considered low, up to 50% moderate, and over 50% high. donors is shown. iDCs were incubated with inhibitors for 45 min at 37 °C and then in the presence or absence of CCL16 for an additional 24 h. Results are expressed as percentage of inhibition for each marker increased or induced by CCL16. One of two experiments 24 h. The isotype control IgG was used as negative control. We is represented. evaluated the expression of CD83, CD80, CD86, CXCR4, and CCR6, mainly increased or induced by CCL16 (Table II). Block- protein G and wortmannin (PI3K) pathways thus control the ade of the CCRs by their specific mAbs differentially affected the CCL16-dependent induction of CCR6 and moderately that of activity of CCL16 on iDCs. The expression of CCR6 seems to be CD86; the PI3K pathway controls CCL16-dependent induction Downloaded from controlled mainly by CCR1, CCR8, and CCR5 because their of CD86; and the p38 MAPK and PLC pathways control CXCR4 blocking abrogated or strongly inhibited the effect of CCL16. Each and CCR6 and partially CD83 and CD86. CCR is equally involved in the induction of the ex novo CXCR4 expression, whereas CCR8 seems more involved in that of CD83. MoDC migration Up-modulation of CD80 and CD86 was slightly inhibited by the We evaluated the ability of CCL16-induced CCR to respond to presence of mAbs. All CCL16’s CCRs would thus seem respon- specific chemokines (Fig. 3), namely CCL4 (Fig. 3A), CCL20 (Fig. http://www.jimmunol.org/ sible for the increased expression of these molecules. 3B), CXCL12 (Fig. 3C), and CCL19 (Fig. 3D). iDCs migrated in To determine which signaling transducing pathway is involved response to CCL4, like CCL16/mDCs, in agreement with their in CCL16-induced maturation of MoDCs, we pretreated iDCs with membrane expression of CCR5. As expected, mDCs did not mi- inhibitors for G protein, PI3K, Erk, p38 MAPK, and phospholipase grate in response to this chemokine. iDCs were not sensitive to C (PLC) pathways at 37°C, followed by incubation in the presence CXCL12 (a CXCR4 agonist) and CCL19 (a CCR7 agonist), which or absence of CCL16 for an additional 24 h. Medium-0.1% DMSO induced migration by both mDCs and CCL16/mDCs. Both types was used as background of stimulation. We evaluated the expres- of mDCs, in fact, express CXCR4 and CCR7 on their surface, sion of CD83, CD86, CCR6, and CXCR4 (Table III). Inhibition of whereas no expression was observed on the iDC membrane. Only the signaling pathways variously affected the activity of CCL16, CCL16/mDCs were sensitive to CCL20 (a CCR6 agonist), a find- by guest on October 1, 2021 with the exception of the Erk pathway. PD98058, in fact, did not ing consistent with their membrane expression of CCR6. Thus, all inhibit CCR expression nor CD83, and only weakly decreased the CCL16-induced chemokine receptors elicited migration to chemo- CCL16-induced CD86 expression. This means that the Erk path- kines expressed by LNs or inflamed peripheral tissues, way is not directly involved in the CCL16-induced maturation of respectively. MoDCs. CCR6 expression was abrogated or strongly inhibited by PTX, wortmannin, SB203580, and U73122. CXCR4 expression Alloactivation by mDCs was abrogated by SB203580 and strongly reduced by LY294002 To check the ability of CCL16/mDCs to present Ag, we analyzed and U73122. Up-modulation of CD86 was differentially inhibited their allostimulatory function because mDCs present alloantigens by the blockade of different pathways: moderately by PTX and and induce proliferation in allogeneic T cells. As shown in Fig. 4A, wortmannin, strongly by LY294002, SB203580, and U73122. CCL16/mDCs and mDCs displayed a similar ability to induce a Lastly, CD83 expression was moderately inhibited by LY234002 significant proliferative response by alloactivated T cells, which and U73122 and strongly by SB203580. Inhibited phosphorylation was higher than that induced by iDCs. Moreover, alloactivated T of different targets was evaluated by Western blot analysis after 45 cells generated with CCL16/mDCs or mDCs showed a similar min of incubation with the inhibitors, followed by 30 min of stim- ability to produce IFN-␥. By contrast, IFN-␥ production by allo- ulation in the presence or not of CCL16 (data not shown). The activated T cells generated in the presence of iDCs was signifi- cantly lower (Fig. 4B). IL-4 was similarly produced by alloacti- Table II. CCR involvement in the CCL16-induced maturation of human vated T cells, irrespective of their generation in the presence of MoDCs iDCs, CCL16/mDCs, or mDCs (Fig. 4C). These data indicate a similar ability of CCL16/mDCs and mDCs to promote a Th1-de- Percentage of Inhibitiona of flected response.

mAb to CD83 CD80 CD86 CCR6 CXCR4 Differential cytokine and chemokine secretion by iDCs, CCL16/mDCs, and mDCs CCR1 25 6 19 100 36 CCR2 14 2 24 33 28 Pools of supernatants of MoDCs from three donors after 48 h of CCR5 35 13 22 60 50 incubation in the absence or presence of CCL16 or TNF-␣ and CCR8 78 15 0 100 46 IL-1␤ were analyzed by the RayBio Human Cytokine Array V to a Inhibition up to 25% is considered low, up to 50% moderate, and over 50% high. simultaneously detect the secretion of 79 factors. After 24 h of iDCs were incubated with anti-CCR mAb for 30 min at 4°C and then in the presence or absence of CCL16 for 24 h. Results are expressed as percentage of inhibition for culture (data not shown), supernatants from CCL16/mDCs pre- each marker increased or induced by CCL16. One of three experiments is represented. sented a limited difference in factor secretion compared with that The Journal of Immunology 6147

FIGURE 3. Activity of CCL16-induced CCRs on MoDCs. iDCs were incubated in medium only (f), or with CCL16 (Ⅺ) or TNF-␣ and IL-1␤ (p) for 24 h before being tested. Medium or different concentrations of chemotactic stimuli were added to cells in a Boyden chamber for an additional2hat37°C. Chemotactic stimuli used: CCL4 (A), CCL20 (B), CXCL12 (C), and CCL19 (D). Each experiment was performed in tripli- cate with three independent donors and represented as p Ͻ 0.05, values ,ء .mean of migrated cells/field Ϯ SD from cells that migrated in the presence of specific che- mokines compared with cells migrated in the absence of stimuli. Downloaded from

by iDCs. They showed a decrease of CCL18 and an increase of CCL20 secretion (data not shown) in agreement with the obser- http://www.jimmunol.org/ vations of other groups (19). After 48 h, several cytokines were increased (Fig. 5A, Ⅺ) by CCL16, including chemokines macro- phage migration inhibitory factor, CCL15, CXCL10, CXCL5, CCL4, and fractalkine. By contrast, another seven factors, GRO, IL-4, CXCL8, IL-10, CCL18, CCL17, and CCL7, were reduced (Fig. 5A, Ⅺ). Six factors, CCL20, IL-1␤, IL-12, CCL23, and IL-2, were induced ex novo by CCL16 and TNF-␣ and IL-1␤ (Fig. 5B). This array provides relative densitometry signals and not absolute by guest on October 1, 2021 concentration values. However, image analysis (UTHSCSA Image Tool for Windows) software, which linearizes OD to spot volume, revealed marked differences in protein secretion between superna- tants from CCL16/mDCs and iDCs (Fig. 5, A and B). For almost all chemokines and cytokines secreted, however, there were no significant differences between supernatants from CCL16/mDCs and mDCs, although GRO, CXCL8, IL-10, CCL2, CXCL5, and IL-6 were decreased by the presence of CCL16. To validate these results, we performed an ELISA for IL-10 and CXCL10, which are inhibited and induced, respectively, by the presence of CCL16, with supernatants from iDCs, CCL16/mDCs, and mDCs from different donors (Fig. 5, C and D). Secretion of both proteins reflected the results obtained with the array. The presence of CCL16 during MoDC maturation, in fact, always de- creased IL-10 secretion (20–454 pg/ml compared with 211–774 pg/ml CCL16/mDCs and iDCs, respectively; Fig. 5C) and in- creased CXCL10 secretion (90–398 pg/ml compared with 17–56 pg/ml CCL16/mDCs and iDCs, respectively; Fig. 5D) in a donor- dependent manner. These data suggest that CCL16/mDCs are not FIGURE 4. CCL16/mDCs induce proliferation and activation of allo- “exhausted.” They secrete many more factors after 48 h of stim- geneic T cells. A, Increasing concentrations of irradiated MoDCs (150– ulation and use them to attract different types of leukocytes. 2400/well) stimulated with medium alone (iDCs, f) or CCL16 (CCL16/ mDCs, E) or TNF-␣ and IL-1␤ (mDCs, Ⅺ) were cultured with 1 ϫ 105 Ag uptake by CCL16/mDCs nylon wool purified T cells for 5 days and for an additional 18 h in the 3 iDCs take up Ag efficiently but lose this ability on maturation (20, presence of [ H]TdR. The results for one of the three donors are shown and p Ͻ 0.02, values from ,ء .represented as mean of counts per minute Ϯ SD 21). Therefore, we measured Ag uptake by iDCs, CCL16/mDCs, T cells cultured with CCL16/mDCs or mDCs compared with T cells cul- and mDCs by flow cytometry (Fig. 6). At 4°C, cell metabolism is tured with iDCs. IFN-␥ (B) and IL-4 (C) production by allogeneic T cells inhibited and such MoDCs are incapable of capturing Ag. How- cultured with iDCs (f), CCL16/mDCs (E), and mDCs (Ⅺ). Six donors ,ء .ever, at 37°C, the control iDCs showed a significant uptake of were tested independently. The horizontal bars mark the mean values FITC-labeled dextran (Fig. 6A) after only 15 min and this progres- p Ͻ 0.05 values from cytokine production by T cells cultured with CCL16/ sively increased after 30 min (Fig. 6, A and D). As expected, mDCs or mDCs compared with that by T cells cultured with iDCs. 6148 MoDC MATURED BY CCL16 CAN STILL TAKE UP Ag Downloaded from http://www.jimmunol.org/

FIGURE 5. Effect of CCL16 during MoDC maturation on cytokine and chemokine production. Pools of three supernatants from iDCs incubated in the absence or presence of CCL16 or TNF-␣ and IL-1␤ for 48 h were analyzed on a RayBio Human Cytokine Array V performed for 79 proteins. The membranes were exposed to x-ray films, analyzed by densitometry, and normalized using the positive controls present in the membranes. Percentage of by guest on October 1, 2021 decrease or increase (A) and the ex novo induction (B) in protein secretion was evaluated by comparing supernatants from CCL16/mDCs (Ⅺ) and mDCs (f) to those from iDCs. N.D., not detected; N.E., not evaluable because present in medium culture. To validate the array results, IL-10 (C) and CXCL10 (D) secretion, inhibited and induced, respectively, by CCL16, were analyzed by ELISA in 12 supernatants from iDCs (f), CCL16/mDCs (E), and from p Ͻ ,ءء ;p Ͻ 0.05 values from protein secretion by CCL16/mDCs compared with that by iDCs ,ء .mDCs (Ⅺ). The horizontal bars mark the mean values 0.05 values from protein secretion by CCL16/mDCs compared with that by mDCs. I.D., intensity of density, as calculated by UTHSCSA image tool for Windows.

mDCs displayed very little ability to take up FITC-labeled dextran the need to use higher amounts of recombinant protein to observe (Fig. 6, C and D). Surprisingly, CCL16/mDCs displayed a high Ag effects. Naturally occurring CCL16 may well be more potent than uptake (Fig. 6B), similar to that of control iDCs after 15 and 30 the recombinant protein. CCL16, moreover, modifies the chemo- min (Fig. 6D). The uptake ability of CCL16/mDCs was maintained kine receptor expression pattern by up-regulating the lymphoid over 60 min (data not shown). chemokine receptors CXCR4 and CCR7 (23, 24) but maintains the presence of the inflammatory CCR5. Interest- Discussion ingly, CCR5 was found on iDCs generated in vitro, whereas mDCs This study demonstrates that CCL16 induces a novel functional have been shown to down-modulate CCR5 and primarily express subset of human MoDCs (CCL16/mDCs), which display a full CCR7 and CXCR4. This is the first feature acquired by CCL16/ maturation phenotype and maintain their ability to take up Ag. We mDCs, even if we did not determine whether CCL16 directly in- evaluated their phenotype, downstream production of chemokines hibits CCR5 degradation or affects its gene regulation. The second and cytokines, and ability to stimulate alloactivated T cells and feature is the induction of CCR6, which is important in the re- take up Ag. CCL16 induces increased levels of MHC class II mol- cruitment of iDCs to the site of injury and typically expressed in ecules and CD80 and CD86 costimulatory molecules and up-reg- CD34ϩ cell-derived DCs and Langerhans cells (25) but not in ulation of the marker CD83 at 100 and 1000 ng/ml. Both concen- MoDCs (26, 27). Furthermore, we demonstrated that CCL16-in- trations induced phenotypic changes in iDCs, but the higher one duced chemokine receptors are functionally active and drive mi- showed greater effects and was used in all the other experiments. gration of CCL16/mDCs in response to specific stimuli. This fea- In a previous report (22), on the other hand, we demonstrated that ture should be very important because CCL16/mDCs could CCL16 secreted by Cos or tumor cells affected endothelial cells activate naive or memory T cells in both inflamed and tumor sites much more efficiently than recombinant protein. Thus, the absence or migrating to LNs. We investigated whether chemokines that of posttranslational modifications in E. coli proteins could explain share the same receptors as CCL16-induced MoDC maturation. The Journal of Immunology 6149

demonstrated a differential CCR1-mediated chemotaxis signaling induced by CCL16 in HOS cells in comparison to other CCR1- dependent chemokines, including CCL3, CCL5, and CCL15, which involves Gi/Go protein, PLC, protein kinase C␥, and p38 MAPK. Studies to better define CCL16 cell transduction in iDCs were performed with several signaling pathway inhibitors. Our preliminary results demonstrate an early and persistent phosphor- ylation of Akt and Erk1–2, even after2hofstimulation (data not shown). The phosphorylation of Akt and Erk1–2 in iDCs after only 10 min of stimulation with CCL16 underlies a direct effect of chemokines on iDCs. However, the Erk pathway seems not to be directly involved in CCL16-induced MoDC maturation since PD98059, a specific MEK inhibitor, did not affect the CD83, CXCR4, and CCR6 expression and only weakly inhibited the up- regulation of CD86. p38 MAPK and PLC are mostly involved as their blockade strongly inhibited the expression of all markers evaluated. Other groups demonstrated that p38 MAPK is prefer- entially involved during the DC maturation induced by LPS or proinflammatory cytokines (30–33). In our case, p38 MAPK path- way is activated by a chemokine. Erk and PI3K have been dem- Downloaded from onstrated to be involved essentially in the DC survival or TNF secretion by LPS-stimulated DCs (30–32). CCL16, however, also FIGURE 6. CCL16/mDCs maintain the ability to take up FITC-dextran. involves PI3K, even if the two inhibitors used displayed differen- MoDCs cultured in medium only (A) or in the presence of CCL16 (B)or tial effects on protein expression by CCL16-stimulated MoDCs. G TNF-␣ plus IL-1␤ (C) for 24 h were further incubated with FITC-dextran proteins, instead, are mainly involved in CCR6 ex novo expres- at 37°C for different times and then analyzed by flow cytometry. A–C, The sion. CCL16 seems able to trigger different pathway signaling to http://www.jimmunol.org/ fluorescence intensity of cells that take up FITC-dextran after 15 min (bold induce DC maturation. Other groups have reported chemokines as line), 30 min (dotted line), and 60 min (thin line). The gray peak shows a inducers of DC activation (34, 35). However, while we show that parallel sample maintained at 4°C with FITC-dextran (negative control). CCL16 affects immature not activated MoDCs, Marsland et al. One of three independent experiments is shown. Referring to the same (35) report that CCL19 and CCL21 are adjuvants for terminal ac- experiment, the difference between the MFI of the sample incubated at tivation of “licensed” or already activated DCs by binding CCR7 37°C and the MFI of that maintained at 4°C was calculated (D) for each time of incubation of iDCs (f), CCL16/mDCs (E), and mDCs (Ⅺ) with expressed only by matured DCs. FITC-dextran. In tumors, a major problem is induction of T cell tolerance by iDCs that present Ag inefficiently. CCL16/mDCs could be very by guest on October 1, 2021 efficient in Ag presentation at the tumor site. There are costimu- latory molecules on their surface and they secrete proteins that Only CCL14, a CCR1 agonist, elicited CD83 and CCR7 expres- favor T cell attraction and activation, such as CCL4 (36), CXCL10 sion, although to a lesser extent than CCL16. We have demon- (37), and CCL13 (38). Molon et al. (39) have demonstrated re- strated that CCL16 induces human monocyte activation especially cently that chemokines secreted by APCs during Ag presentation through binding with CCR1 (28), and we supposed that even are sufficient to recruit chemokine receptors on the T cell mem- MoDCs could be affected by CCL16-CCR1 binding. However, brane at the immunological synapse. This trapping enhances T cell neutralizing mAbs to each CCL16 receptor, when added to iDCs proliferation and cytokine production (39). CCL16/mDCs show during a chemotaxis assay, inhibited iDC migration, suggesting the same ability as classic mDCs to prime T cells to mismatched that they are functional receptors mediating CCL16’s effects on MHC. CCL16/mDC-alloactivated T cells produced increased lev- iDC migration. Interestingly, combined treatment of iDCs with els of IFN-␥ compared with those activated in the presence of neutralizing mAbs did not further inhibit their migration in re- iDCs. CCL16/mDCs thus prime and activate Th1 cells. sponse to CCL16 compared with the single mAb alone. These data The ability of CCL16/mDCs to take up Ag, compared with the suggest that all CCRs are used by CCL16, perhaps cooperatively, inability of classic mDCs, is their third interesting feature. Usually to stimulate iDC migration. We cannot determine from these ex- iDCs are less potent initiators of immunity but specialized in cap- periments whether the receptors interact physically or simply co- turing and processing Ags to form MHC peptide complexes. DCs operate. When we investigated which receptor is used by CCL16 thus perform their two key functions at different times and in dif- in iDC maturation by pretreatment of iDCs with neutralizing ferent locations: they first handle Ags as iDCs, and then, as mDCs, mAbs, we found that all receptors are differently involved. The stimulate T cells in LNs (40). This prolonged ability to take up Ag expression of CCR6 is induced mainly by CCL16 binding to could be important to offer Ag for T cell recognition and activation CCR1, CCR8, and CCR5 because their blocking by specific mAbs by subsequent challenge of their TCR (41) at both the tumor and abrogated or strongly inhibited CCL16 effects. Each CCR is inflamed sites, or in LNs. equally involved in the induction of ex novo CXCR4 expression, CCL16/mDCs are still sensitive to further maturation stimuli. In whereas CCR8 is more involved in that of CD83. Costimulatory the presence of TNF-␣ and IL-1␤, they increase CD83 expression molecule expression was only slightly inhibited by each of the and are no longer able to take up FITC-dextran (data not shown). mAbs to CCRs. This result suggests that all receptors cooperate to This suggests that CCL16 prevents what has been called “DC ex- induce their expression or that other chemokines induced by haustion”: after activation by LPS, DCs produce cytokines, espe- CCL16 can cooperate with it. Altogether, these data indicate that cially IL-12, only transiently and became refractory to further CCL16 induces different signaling cascades leading to a “new” stimulation, including CD40L (42). Transient cytokine production maturation program by binding different receptors. Kim et al. 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