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Human Tolerogenic Dendritic CellsProtein Kinase C Inhibitor Generates Stable

Ishizaki, Koichiro Suemori and Masaki YasukawaTakuya Matsumoto, Hitoshi Hasegawa, Sachiko Onishi, Jun

http://www.jimmunol.org/content/191/5/2247doi: 10.4049/jimmunol.1203053July 2013;

2013; 191:2247-2257; Prepublished online 22J Immunol 

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3.DC1http://www.jimmunol.org/content/suppl/2013/07/22/jimmunol.120305

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The Journal of Immunology

Protein Kinase C Inhibitor Generates Stable HumanTolerogenic Dendritic Cells

Takuya Matsumoto,* Hitoshi Hasegawa,*,† Sachiko Onishi,* Jun Ishizaki,*

Koichiro Suemori,* and Masaki Yasukawa*,†

Tolerogenic dendritic cells (DCs) are a promising tool for a specific form of cellular therapy whereby immunological tolerance can be

induced in the context of transplantation and autoimmunity. From libraries of bioactive lipids, nuclear receptor ligands, and kinase

inhibitors, we screened conventional protein kinase C inhibitors (PKCIs) bisindolylmaleimide I, Go6983, andRo32-0432 with strong

tolerogenic potential. PKCI-treated human DCs were generated by subjecting them to a maturation process after differentiation of

immature DCs. The PKCI-treated DCs had a semimature phenotype, showing high production of IL-10, and efficiently induced IL-

10–producing T cells and functional Foxp3+ regulatory T cells from naive CD4+ T cells, thus eliciting a strong immunosuppressive

function. They also showed CCR7 expression and sufficient capacity for migration toward CCR7 ligands. Additionally, PKCI-

treated DCs were highly stable when exposed to inflammatory stimuli such as proinflammatory cytokines or LPS. Conventional

PKCIs inhibited NF-kB activation of both the canonical and noncanonical pathways of DC maturation, thus suppressing the

expression of costimulatory molecules and IL-12 production. High production of IL-10 in PKCI-treated DCs was due to not only

an increase of intracellular cAMP, but also a synergistic effect of increased cAMP and NF-kB inhibition. Moreover, PKCI-treated

mouse DCs that had properties similar to PKCI-treated human DCs prevented graft-versus-host disease in a murine model of acute

graft-versus-host disease. Conventional PKCI-treated DCs may be useful for tolerance-inducing therapy, as they satisfy the re-

quired functional characteristics for clinical-grade tolerogenic DCs. The Journal of Immunology, 2013, 191: 2247–2257.

Dendritic cells (DCs) are a heterogeneous population ofAPCs that contribute to innate immunity and initiate,coordinate, and regulate adaptive immune responses in

infection and inflammation (1). Successful initiation of theseadaptive immune responses requires DC maturation. Immature DCs(iDCs) are able to take up soluble Ag but are poorly immunogenicbecause they express only modest levels of MHC molecules and lowlevels of costimulatory molecules and proinflammatory cytokines.During maturation, DCs lose their capacity to capture soluble Agsbut gain T cell stimulatory capacity due to increased Ag processingand upregulation of MHC, costimulatory molecules, and cytokines(1, 2). Although potently capable of initiating adaptive immuneresponses, DCs also play an important role in modulating the in-duction of tolerance (3–5). Tolerogenic DCs (tDCs) are generallycharacterized by an immature or semimature phenotype, with a ca-pacity for high Ag uptake and low expression of costimulatorymolecules (2, 6). Additionally, tDCs produce low amounts of

proinflammatory cytokines and high amounts of anti-inflammatorycytokines. This results in anergy, apoptosis of effector T cells, orinduction and expansion of regulatory T cells (7).For patients with autoimmune diseases and graft rejection, any

therapy capable of preventing or reducing the extant immune ac-tivation is highly desirable. Ex vivo–generated tDCs are consideredto have strong potential for use in cellular therapy for these con-ditions. In fact, injection of ex vivo–generated tDCs has beenshown to be beneficial in animal models of graft rejection (8–10)and autoimmune diseases, including collagen-induced arthritis (11,12), diabetes (13–16), and experimental autoimmune enceph-alomyelitis (17). Human tDCs can be cultured in vitro from DCprecursors using different compounds (7, 18). For this purpose,anti-inflammatory cytokines such as IL-10 (19–22) and TGF-b(22–24), vitamin D3 (Vit D3) (25–28), neuropeptides, includingvasoactive intestinal peptide (17, 29, 30) and a-melanocyte–stim-ulating hormone (31), and anti-inflammatory/immunosuppressivedrugs such as dexamethasone (Dexa) (28, 32, 33) and rapamycin(Rapa) (34–36) are often employed. However, the compound mostsuitable for clinical application remains undefined from the view-point of tolerogenic potential, stability, and capacity for migrationtoward secondary lymphoid organs, that is, the area where immuneresponses occur (37–39). Therefore, to obtain compounds that havestronger tolerogenic potential, we screened libraries of bioactivelipids, nuclear receptor ligands, and kinase inhibitors for moleculesthat would downregulate the costimulatory molecules CD80 andCD86 on DCs and produce the highest level of IL-10. Conse-quently, we obtained some protein kinase C inhibitors (PKCIs).Members of the PKC family are serine-threonine protein kinases

that are divided into three subfamilies on the basis of their substratespecificity: conventional a, bΙ, bΙΙ, and g PKCs are diacylglycerol(DAG)- and calcium-dependent; novel ε, d, m, u, and h PKCs areDAG-dependent and calcium-independent; and atypical z, i, and lPKCs respond to neither DAG nor calcium (40). It has been re-ported that modulation of cellular individual PKC activity induces

*Department of Bioregulatory Medicine, Ehime University Graduate School of Med-icine, Toon, Ehime 791-0295, Japan; and †Proteo-Medicine Research Center, EhimeUniversity Graduate School of Medicine, Toon, Ehime 791-0295, Japan

Received for publication November 5, 2012. Accepted for publication June 18, 2013.

This work was supported in part by grants-in-aid for scientific research from theMinistry of Education, Culture, Sports, Science and Technology of Japan.

Address correspondence and reprint requests to Dr. Hitoshi Hasegawa, Department ofBioregulatory Medicine, Ehime University Graduate School of Medicine, Toon,Ehime 791-0295, Japan. E-mail address: hitoshih@m.ehime-u.ac.jp

The online version of this article contains supplemental material.

Abbreviations used in this article: Bis I, bisindolylmaleimide I; BM, bone marrow;BMT, bone marrow transplantation; DAG, diacylglycerol; DC, dendritic cell; Dexa,dexamethasone; GVHD, graft-versus-host disease; iDC, immature dendritic cell;KRH, Krebs–Ringer HEPES; mDC, mature dendritic cell; MMC, mitomycin C; noAbs-C, without anti–IL-10 and anti–TGF-b mAb; PKC, protein kinase C; PKCI,protein kinase C inhibitor; Rapa, rapamycin; rh, recombinant human; rm, recombi-nant mouse; tDC, tolerogenic dendritic cell; Vit D3, vitamin D3.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

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PKC-dependent cell differentiation and cytokine production inmany cell types (40). PKC has been reported to play critical rolesin mediating progenitor-to-DC differentiation and cytokine pro-duction from monocytes/macrophages and DCs (41–46). In thepresent study, we found that stable tDCs showing high productionof IL-10 were generated by addition of PKCI during the process ofmaturation from iDCs. We also characterized the PKCI-treatedtDCs obtained and clarified the mechanism responsible for theirinduction.

Materials and MethodsAbs and reagents

FITC-conjugated mouse anti-human CD1a (HI149), PE-conjugated mouseanti-human CD11c (B-ly6), allophycocyanin-conjugated mouse anti-humanCD4 (RPA-T4), allophycocyanin-conjugated mouse anti-human CD14(MfP9), and PE-conjugated rat anti-human IL-10 (JES3-19F1) mAbs werepurchased from BD Biosciences (San Diego, CA). PE-conjugated mouseanti-human CD40 (MAB89), FITC-conjugated mouse anti-human CD80(MAB104), FITC-conjugated mouse anti-human CD83 (HB15a), PE-conjugated mouse anti-human CD86 (HA5.2B7), FITC-conjugated mouseanti-human HLA-ABC (B9.12.1), and PE-conjugated mouse anti-humanHLA-DR (E8.12.2) mAbs were obtained from Beckman Coulter (Brea,CA). PE-conjugated rat anti-human Foxp3 (PCH101) and mouse anti-human CCR7 (CCR7.6B3) (47) mAbs were obtained from eBioscience(San Diego, CA). FITC-conjugated mouse anti-human CD25 (BC96) mAbwas perchased from BioLegend (San Diego, CA). Recombinant human (rh)GM-CSF, rhIL-4, rhIL-1b, rhIL-6, rhCCL19, mouse anti-human IL-10(25209), and mouse anti-human TGF-b1 (9016) mAbs were obtainedfrom R&D Systems (Minneapolis, MN). IFN-g was purchased fromPeproTech (Rocky Hill, NJ). rhTNF-a was obtained from DainipponPharmaceutical (Osaka, Japan). Five kinds of PKCIs—bisindolylmaleimideI (Bis I) (48), Go6983 (49), Ro32-0432 (50), Go6976, and HBDDE—andlibraries of bioactive lipids, nuclear receptor ligands, and kinase inhibitorswere purchased from Enzo Life Sciences (Plymouth Meeting, PA). CGP53353was obtained from Tocris Bioscience (Bristol, U.K.). Bis I, Go6983, andRo32-0432 are conventional PKCIs, and all share a common inhibitionof a, b, and g isoforms (48–50). Go6976, CGP53353, and HBDDE arePKCa- and bI-specific, PKCbII-specific, and PKCa- and g-specific inhibitors,respectively. PGE2 and LPS were obtained from Sigma-Aldrich (St.Louis, MO).

FITC-conjugated hamster anti-mouse CD11c (HL3), FITC-conjugatedrat anti-mouse CD40 (3/23), FITC-conjugated hamster anti-mouse CD80(16-10A1), PE-conjugated rat anti-mouse CD83 (Michel-19), FITC-conjugated rat anti-mouse CD86 (GL1), PE-conjugated mouse anti-mouse H-2Kd (SF1-1.1), and PE-conjugated rat anti-mouse I-A/I-E(2G9) mAbs were purchased from BD Biosciences. Carboxyfluorescein-conjugated rat anti-mouse CCR7 (4B12) mAb, recombinant mouse (rm)GM-CSF, rmTNF-a, rmIL-1b, and rmCCL19 were obtained from R&DSystems.

Generation of human DCs

Human peripheral blood was obtained from healthy donors after obtaininginformed consent in accordance with procedures approved by the HumanEthics Committee, Ehime University. CD14+ cells from PBMCs were iso-lated using a CD14 isolation kit (Miltenyi Biotec, Bergisch Gladbach,Germany). The purity of the isolated CD14+ monocytes was .95% uponflow cytometric analysis. iDCs were generated from the CD14+ monocytesby culturing them in RPMI 1640 medium supplemented with 10% FBS(Life Technologies, Rockville, MD), 75 ng/ml rhGM-CSF, and 10 ng/mlrhIL-4 for 5 d. To induce maturation (mature DCs [mDCs]), iDCs wereincubated with a maturation mixture containing rhTNF-a (10 ng/ml), rhIL-1b (10 ng/ml), and PGE2 (1 mg/ml) for a further 48 h. PKCI-treated DCswere generated by culturing iDCs with a maturation mixture in the presenceof PKCIs such as Bis I (10 mM), Go6983 (10 mM), or Ro32-0432 (10 mM)for 48 h.

Flow cytometric analysis

DC cell surface expression and the expression of Foxp3 and IL-10 in CD4+

T cells were analyzed using each of the mAbs described in the Abs andreagents section labeled with FITC or PE on a FACSCalibur flow cy-tometer (BD Biosciences), as described previously (51). For intracellularFoxp3 and IL-10 staining, permeabilization was performed in accordancewith the manufacturer’s protocol.

Assay for cytokine production from DCs

iDCs, mDCs, and PKCI-treated DCs were adjusted to 1 3 106 per well in1 ml and cultured in RPMI 1640 medium supplemented with 10% FBS forassay of IL-6, IL-10, IL-12, and IFN-g, or in X-VIVO medium only forassay of TGF-b. The cell supernatants were harvested after 96 h, con-taminating cells were removed by centrifugation, and the supernatantswere frozen at 270˚C until used. The supernatants were then assayedusing a sandwich ELISA (R&D Systems) (IL-6, IL-10, IL-12, IFN-g, andTGF-b), in accordance with the manufacturer’s instructions.

Phagocytic ability

Phagocytic ability was measured as the cellular uptake of FITC/latex beads/rabbit IgG using a phagocytosis assay kit (Cayman Chemical, Ann Arbor,MI). DCs were incubated with FITC/latex beads/rabbit IgG for 24 h ina CO2 incubator and then analyzed by flow cytometry in accordance withthe manufacturer’s instructions.

Stability of PKCI-treated DCs

Phenotypic stability of PKCI-treated DCs was determined in response toinflammatory stimuli, as described by Harry et al. (37). Briefly, PKCI-treated DCs, iDCs, or mDCs were washed, adjusted to 5 3 105 per wellin 1 ml, and cultured in RPMI 1640 medium supplemented with 10% FBSor in X-VIVO medium only for assay of TGF-b in the absence or presenceof a mixture of proinflammatory cytokines containing IFN-g at 1000 U/ml,IL-1b, IL-6, and TNF-a each at 10 ng/ml, or LPS at 0.1 mg/ml. After 1 and5 d, DCs were analyzed for cell surface phenotype by flow cytometry, andproduction of IL-10 and TGF-b in the culture supernatants was determinedby ELISA. To examine the functional properties of the DCs, they werewashed after stimulation with a mixture of proinflammatory cytokines orLPS and recultured in RPMI 1640 medium supplemented with 10% FBSfor a further 24 h. Then, an MLR was performed as described in the invitro T cell proliferation assay section.

In vitro T cell proliferation assay

Human CD4+ T cells were isolated from PBMCs using a CD4+ T cellisolation kit II (Miltenyi Biotec). Freshly isolated CD4+ T cells (1 3 105)were cocultured with 1 3 104 (10:1 T cell/DC ratio) or 2 3 104 (10:2)mitomycin C (MMC)–treated allogeneic DCs in a final volume of 200 mlX-VIVO medium supplemented with 10% FBS in 96-well round-bottomplates for 4 d. The wells were pulsed with 1 mCi [3H]thymidine (AmershamBiosciences, Piscataway, NJ) 18 h before harvesting.

For mouse in vitro T cell proliferation assays, mouse CD4+ T cells wereisolated from spleens of C57BL/6 mice using a CD4+ T cell isolation kit(Miltenyi Biotec). MMC-treated allogeneic DCs were prepared fromBALB/c mice.

Induction of regulatory T cells by PKCI-treated DCs

Induction of human regulatory T cells by PKCI-treated DCs was performedas described by Sato et al. (21). Briefly, human naive CD4+CD45RA+

T cells were purified from PBMCs with a CD4+ T cell isolation kit II(Miltenyi Biotec) and subsequent negative selection with anti-CD45ROmAb-conjugated immunomagnetic beads (Miltenyi Biotec). Human na-ive CD4+CD45RA+ T cells (5 3 106) were cultured with 5 3 105 MMC-treated allogeneic iDCs, mDCs, or PKCI-treated DCs in X-VIVO mediumwith 10% FBS for 5 d. Then, Foxp3+CD4+CD25+ T cells and IL-10+CD4+

T cells were analyzed by flow cytometry.

In vitro T regulatory activity

In vitro T regulatory activity was modified as described previously (51) andas reported by Gonzalez-Rey et al. (30). Human Th1 cells were generatedfrom naive CD4+CD45RA+ T cells. Briefly, naive CD4+CD45RA+ T cells(1 3 106) were cultured with 1 3 105 MMC-treated allogeneic mDCs inX-VIVO medium with 10% FBS. Alloreactive T cells were expanded fromday 6 in the presence of 100 U/ml rhIL-2. Two weeks after priming, T cellswere restimulated with mDCs from the same donor under identical cultureconditions. CD4+ T cells were isolated 1 wk after restimulation and used asalloreactive Th1 cells.

Human naive CD4+CD45RA+ T cells (5 3 106) were cultured with 5 3105 MMC-treated allogeneic DCs treated with PKCI in X-VIVO mediumwith 10% FBS for 5 d. Then, CD4+ T cells were purified from the abovecocultures with a CD4+ T cell isolation kit II (Miltenyi Biotec). These pu-rified CD4+ T cells (1 3 105) were incubated with syngeneic Th1 cells(responder T cells, 1 3 105) in the presence of MMC-treated allogeneicmDCs (13 104) in a final volume of 200 ml X-VIVO medium supplementedwith 10% FBS in 96-well round-bottom plates for 4 d. The proliferation of

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responder T cells was determined by [3H]thymidine incorporation. In someexperiments, cocultures were performed in the presence of blocking mouseanti–IL-10 (40 mg/ml) and/or anti–TGF-b1 (40 mg/ml) mAbs. The anti–IL-10 and anti–TGF-b1 mAbs had been titrated previously. Concentrationsresulting in undetectable levels of IL-10 or TGF-b1 following Ab additionwere used in the blocking experiments.

Transwell experiments were performed using 24-well plates (Millicell,0.4 mm; Millipore, Bedford, MA). Th1 cells (5 3 105) together with MMC-treated allogeneic mDCs (5 3 104) were placed in the bottom wells.CD4+ T cells (5 3 105) generated in the presence of PKCI-treated DCswere placed in the upper wells. The cultures were performed in the absenceor presence of anti–IL-10 and/or anti–TGF-b1 mAbs for 4 d. Then, thebasket was removed and the proliferation of the responder T cells wasmeasured.

ELISA for measurement of RelA and RelB

Human iDCs (1 3 106/well) were incubated with PKCI for 2 h and thenstimulated with TNF-a (10 ng/ml), IL-1b (10 ng/ml), and/or PGE2 (1 mg/ml).After 4 h of stimulation, nuclear and cytoplasmic extracts from DCs wereseparated using a nuclear extraction kit (Imgenex, San Diego, CA). Acti-vated NF-kBs (RelA or RelB) in the nuclear extracts were measured byoligonucleotide-based ELISA as described by Bhattacharya et al. (52).Briefly, the extracts from DCs were incubated for 1 h at room temperaturewith agitation in wells of a 96-well microtiter plate that had been coatedwith the NF-kB consensus nucleotide sequence (59-GGGACTTTCC-39).NF-kB from the cell samples attached to the wells was captured by an Abspecific to either RelA or RelB (Santa Cruz Biotechnology, Santa Cruz,CA). Binding of the specific NF-kB family member was then detectedusing an anti-rabbit HRP-conjugated IgG. Color was developed with hy-drogen peroxide/tetramethylbenzidine chromogenic substrate, and inten-sity of the developed color was proportional to the quantity of RelA andRelB in each sample. The sample values were normalized against total cellprotein determined using a protein assay kit (Pierce, Rockford, IL).

cAMP assay

A cAMP assay was performed as described previously (53). After humaniDCs (23 105/well) had been cultured in a 96-well plate for 12 h, the cellswere incubated with PKCIs for 1 h. The cells were then washed twice withKrebs–Ringer HEPES (KRH) buffer (1.24 mM NaCl, 5 mM KCl, 1.25 mMMgSO4, 1.45 mM CaCl2, 1.25 mM KH2PO4, 25 mM HEPES [pH 7.4], and8 mM glucose), 90 ml KRH buffer containing 10 mM forskolin (Sigma-Aldrich) with or without PKCI was added, and then the cells were stim-ulated with TNF-a, IL-1b, and/or PGE2 for 1 h. The cells were lysed byadding 200 ml lysis buffer 1A (a component of the cAMP enzyme im-munoassay system; Amersham Biosciences), and 100 ml cell lysate wasused for measurement of cAMP produced during incubation with the kit,as recommended by the manufacturer.

Chemotaxis assay

Chemotaxis assays for iDCs, mDCs, and PKCI-treated DCs were performedin polycarbonate-membrane, 6.5-mm-diameter, 5-mm-pore size Transwellcell culture chambers (Costar, Cambridge, MA), as described previously(54). Aliquots (100 ml) of cells (53 106/ml) suspended in RPMI 1640/0.5%BSA were added to the upper chambers. The CCL19 was added to thelower wells at a concentration of 1 mg/ml. The cells were allowed tomigrate for 2 h at 37˚C in a 5% CO2 incubator, after which the filterswere fixed with 1% glutaraldehyde in PBS for 30 min and stained with0.5% toluidine blue overnight. Cell migration was quantified by countingthe cells in each lower chamber and cells adhering to the bottom of thepolycarbonate filter.

PKC kinase activity

iDCs, mDCs, and PKCI-treated DCs (1 3 106) at various PKCI concen-trations were lysed with lysis buffer (20 mM MOPS, 50 mM b-glycer-ophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mMEGTA, 2 mM EDTA, 1% Nonidet P-40, 1 mM DTT, 1 mM benzamidine,1 mM PMSF, and 10 mg/ml leupeptin and aprotinin). After centrifugation,each supernatant was analyzed for PKC activity using a PKC kinase activitykit (Enzo Life Sciences), in accordance with the manufacturer’s protocol.

Mice

Female BALB/c (H-2d) and C57BL/6 (H-2b) mice were purchased fromClea Japan (Tokyo, Japan). The graft-versus-host disease (GVHD) exper-iment was approved by the Aminal Ethics Committee, Ehime University.The mice used for the GVHD experiment ranged from 10 and 14 wk ofage. They were housed in sterilized microisolator cages after bone marrow

transplantation (BMT) and received filtered water and autoclaved chow orautoclaved drinking water.

Generation of mouse DCs

Mouse iDCs, mDCs, and PKCI-treated DCs were generated from BM cellsas described by Chorny et al. (55). Briefly, BM cells (2 3 106) obtainedfrom BALB/c mice were incubated in RPMI 1640 medium with 10% FBSand 20 ng/ml rmGM-CSF for 6 d. Then, nonadherent cells (iDCs) werecollected (routinely comprising 80–90% CD11c+ cells) and stimulated withLPS (1 mg/ml) or a maturation mixture containing rmTNF-a (50 ng/ml),rmIL-1b (10 ng/ml), and PGE2 (200 ng/ml) for 48 h to induce maturation.PKCI-treated DCs were generated by culturing iDCs with a maturationmixture in the presence of Bis I (10 mM) for 48 h.

BMT and GVHD induction

BMT and GVHD induction were performed as described previously (54)and by the method of Chorny et al. (55). Briefly, recipients (BALB/c)received 10 Gy total body irradiation, split into two doses separated by12 h to minimize gastrointestinal toxicity. Donor BM (C57BL/6) was re-moved aseptically from femurs and depleted of T cells with anti-mouseThy 1.2 mAb (Acris Antibodies, San Diego, CA) and Low-Tox-M rabbitcomplement (Cedarlane Laboratories, Hornby, ON, Canada). SplenicT cells were prepared by purification over nylon wool for GVHD induc-tion. T cell–depleted BM cells (1.5 3 107) plus 2 3 106 splenic T cellsfrom allogeneic (C57BL/6) donors were resuspended in 0.2 ml PBS andinjected i.v. into recipients (BALB/c) on day 0 for induction of acuteGVHD. Recipients received a single i.v. injection of host-matched iDCs,mDCs, or PKCI-treated DCs (BALB/c, 2 3 106) 2 d after transplantation.Recipients were monitored daily from the day of transplantation until theydied naturally of GVHD.

Statistical analysis

Data are expressed as means6 SD. One-way ANOVAwas used to comparemultiple groups. When the ANOVA indicated significance, the Tukey–Kramer honestly significant difference test was applied for multiplecomparisons. Differences in survival of treatment groups in the GVHDexperiment were analyzed using Kaplan–Meier and log-rank tests. Ap value of ,0.05 was considered to indicate statistical significance.

ResultsCharacterization of PKCI-treated human DCs

PKCI-treated human DCs were prepared by culturing iDCs witha maturation mixture (TNF-a, rhIL-1b, and PGE2) for 48 h in thepresence of a conventional PKCIs such as Bis I, Go6983, or Ro32-0432. In the preliminary experiment, the optimal concentrationof three PKCI compounds, Bis I, Go6983, and Ro 32-0432, wasdecided from downregulation of costimulatory molecules (CD40,CD80, and CD86), viability, and inhibition of PKC activity. Amongthe concentrations of 1, 10, and 30 mM, 10 mM was the optimal onefor all 3 PKCIs, resulting in strong downregulation of costimulatorymolecules, a viability of .95%, and inhibition of PKC activity thatreached a plateau (Supplemental Fig. 1). Therefore, we used 10 mMfor all PKCIs in this study.First, we compared the surface phenotypes of iDCs, mDCs, and

PKCI-treated DCs. As shown in Fig. 1A, iDCs showed moderatelevels of expression of CD40 and MHC class I, low expression levelsof CD11c, CD80, CD86, and MHC class II, and extremely lowlevels of CD1a and CD83. After inducing maturation with a matu-ration mixture (mDCs), the expression levels of CD40, CD80, CD83,CD86, MHC class I, and MHC class II were increased significantlycompared with those of iDCs. DCs treated with PKCIs such as Bis I,Go6983, and Ro32-0432 showed significantly decreased levels ofexpression of CD40, CD80, CD83, CD86, and MHC class I, butnot CD1a, CD11c, and MHC class II, in comparison with mDCs.CD14, a monocyte marker, was expressed slightly on iDCs but noton mDCs or PKCI-treated DCs (Fig. 1B).Because migration of DCs to the T cell zone of secondary

lymphoid organs is guided by the homing chemokines CCL19 andCCL21 (56), we examined the expression of CCR7, their receptor

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on iDCs, mDCs, and PKCI-treated DCs. As shown in Fig. 1A,barely any expression of CCR7 was evident on iDCs. In contrast,upregulation of CCR7 expression was recognized on mDCs andPKCI-treated DCs, although the expression level of CCR7 onPKCI-treated DCs was lower than that on mDCs. Functionally, thecapacity of PKCI-treated DCs to migrate toward CCL19 was ∼70–80% of that of mDCs (no significant difference versus mDCs),whereas iDCs showed little or no migration (Fig. 1C).Next, we examined cytokine production of iDCs, mDCs, and

PKCI-treated DCs. Cytokines in the 96-h culture supernatants ofiDCs, mDCs, and PKCI-treated DCs were measured. As shown inFig. 2A, after stimulation with a maturation mixture, iDCs ma-tured into cells capable of producing IL-12, but with reducedproduction of IL-10 and TGF-b. In contrast, PKCI-treated DCsproduced a 15- to 20-fold higher concentration of IL-10 and a 3-to 5-fold higher concentration of TGF-b than did mDCs. How-ever, the production of IL-12 by PKCI-treated DCs was muchlower than that by mDCs.We then examined the ability of iDCs, mDCs, and PKCI-treated

DCs to induce the proliferation of allogeneic CD4+ T cells. Asshown in Fig. 2B, iDCs reduced the proliferation of allogeneicCD4+ T cells significantly by 25 6 10 and 48 6 10% at a ratio of1:10 and 2:10 (DC/T cell), respectively, in comparison with

mDCs. Furthermore, the degree of PKCI-treated DC-inducedT cell hyporesponsiveness was higher than that of iDCs (a ratioof 1:10 and 2:10 [DC/T cell]: Bis I, 69 6 4 and 89 6 10%;Go6983, 77 6 8 and 83 6 8%; and Ro32-0432, 79 6 5 and 78 610%; p , 0.01). PKCI-treated DCs retained their hypores-ponsiveness for at least 1 wk. Therefore, this hyporesponsivenessof PKCI-treated DCs was mainly due to downregulation of costim-ulatory molecules CD80, CD83, and CD86 and high productionof IL-10 and TGF-b.We assessed the phagocytic capacity of PKCI-treated DCs using

FITC/latex beads/rabbit IgG. As shown in Fig. 2C, DCs lost theirphagocytic ability during maturation. However, PKCI-treated DCsretained their phagocytic ability, similar to iDCs.For clinical grade DC preparation, iDCs, mDCs, and PKCI-

treated DCs were generated using serum-free X-VIVO 15 me-dium instead of RPMI 1640 medium plus 10% FBS. The iDCs,mDCs, and PKCI-treated DCs prepared in X-VIVO 15medium hadproperties similar to those of cells prepared in RPMI 1640 mediumwith 10% FBS (Supplemental Figs. 2, 3).

FIGURE 1. PKCI suppresses expression of costimulatory molecules on

human DCs. (A) Surface phenotype on iDCs, mDCs, and PKCI-treated DCs.

(B) CD14 expression on monocytes, iDCs, mDCs, and PKCI-treated DCs.

PKCI-treated DCs were prepared by culturing iDCs with a maturation

mixture in the presence of PKCIs such as Bis I (10 mM), Go6983 (10 mM),

or Ro32-0432 (10 mM) for 48 h. Surface phenotype on DCs was analyzed by

flow cytometry. Numbers in the corners indicate the mean fluorescence in-

tensity (MFI) of expression level of each surface marker. Representative data

from three experiments are shown. (C) Chemotactic activity of iDCs, mDCs,

and PKCI-treated DCs to CCL19. Aliquots (100 ml) of cells (5 3 106/ml)

were added to the upper chambers, and CCL19 (1 mg/ml) was added to the

lower wells. After 2 h, cell migration was quantified by counting cells in each

lower chamber and cells adhering to the bottom part of the filter. Each assay

was performed in triplicate. *p , 0.01.

FIGURE 2. Characterization of human iDCs, mDCs, and PKCI-treated

DCs. (A) Cytokine production from iDCs, mDCs, and PKCI-treated DCs.

iDCs, mDCs, and PKCI-treated DCs were prepared as described in Mate-

rials and Methods. These DCs (1 3 106/ml) were cultured in RPMI 1640

medium supplemented with 10% FBS for assay of IL-6, IL-10, IL-12, and

IFN-g, or in X-VIVO medium only for assay of TGF-b. The cell super-

natants were harvested after 96 h and assayed. Values are the means 6 SD

of three experiments. *p , 0.01. (B) Comparison of suppressive properties

among iDCs, mDCs, and PKCI-treated DCs. Freshly isolated CD4+ T cells

(1 3 105) were cocultured with 1 3 104 (10:1 T cell/DC ratio) or 2 3 104

(10:2) MMC-treated allogeneic iDCs, mDCs, and PKCI-treated DCs. After

4 d in culture, T cell proliferation was determined using [3H]thymidine

incorporation. T cell proliferation by iDCs and PKCI-treated DCs was ex-

pressed relative to that by mDCs. Values are the means 6 SD (n = 6/ex-

periment). Representative data from three experiments are shown. *p ,0.05, **p , 0.01. (C) Phagocytic ability of iDCs, mDCs, and PKCI-treated

DCs. DCs were incubated with FITC/latex beads/rabbit IgG for 24 h and

then analyzed by flow cytometry.

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PKCI-treated human DCs induce IL-10–producing T cells andfunctional regulatory T cells

Because PKCI-treated DCs produced IL-10 and TGF-b, we ex-amined whether they would induce IL-10–producing T cells andFoxp3+CD4+CD25+ T cells. Naive CD4+CD45RA+ T cells werecocultured with iDCs, mDCs, or PKCI-treated DCs for 5 d, andthen intracellular IL-10+CD4+ T cells and Foxp3+CD4+CD25+

T cells were analyzed by flow cytometry. As shown in Fig. 3A,PKCI-treated DCs generated .4-fold the number of both IL-10+

CD4+ T cells and Foxp3+CD4+CD25+ T cells than did those in-duced by mDCs. CD4+ T cells cocultured with PKCI-treated DCssuppressed the proliferation of effector Th1 cells most strongly(Bis I, 48 6 4%; Go6983, 41 6 14%; and Ro32-0432, 36 6 6%),and CD4+ T cells cocultured with iDCs did so to a lesser degree(26 6 7%), compared with control (Fig. 3B).Next, we addressed the question of whether this suppression was

due to direct cellular contact and/or soluble factors. In the cocultureexperiments, addition of saturating amounts of anti–IL-10 or anti–TGF-b1 mAbs reversed the inhibition only moderately (percent-age suppression of effector Th1 proliferation without anti–IL-10and anti–TGF-b mAbs (no Abs-C), with only anti-IL-10 mAb,and with only anti-TGF-b mAb: 48 6 4, 32 6 8, and 30 6 5%,respectively). Addition of both anti–IL-10 and anti–TGF-b1mAbs to the cocultures reversed the inhibition more markedly,although the inhibition still persisted (21 6 9%). When effectorTh1 cells and CD4+ T cells cocultured with Bis I–treated DCswere separated in the Transwell experiments, the proliferation ofeffector Th1 cells was still inhibited to a lesser degree (21 6 5%).Addition of both anti–IL-10 and anti–TGF-b1 mAbs in the Trans-well experiments reversed the inhibitory effect almost completely(Fig. 3C). Similar results were obtained from the experiments usingCD4+ T cells cocultured with DCs treated with two other PKCIs,Go6983 and Ro32-0432 (data not shown). These findings indi-cated that both direct contact and soluble factors mediate theinhibitory effect of CD4+ T cells cocultured with PKCI-treatedDCs. Additionally, it was shown that the immunosuppressivefunction of PKCI-treated DCs was elicited by generating IL-10–producing T cells and functional Foxp3+ regulatory T cells fromnaive CD4+ T cells.

Tolerogenic properties of PKCI-treated human DCs are highlystable

A potential risk of ex vivo–generated tDCs is that they may switchto an activating phenotype after encountering danger signalsin vivo. To address this issue, the stability of PKCI-treated DCswas examined by stimulation with proinflammatory mediators suchas proinflammatory cytokines and LPS, after removal of PKCI. Wecompared the surface phenotype of DCs, cytokine production, andT cell proliferation on the first day and the fifth day after stimu-lation with proinflammatory mediators. As shown in Fig. 4, theexpressions of CD80, CD83, CD86, and MHC class II on PKCI-treated DCs were upregulated only minimally and remained low,even on the fifth day after stimulation. Additionally, CCR7 ex-pression on PKCI-treated DCs after stimulation kept the similarlevel to unstimulated PKCI-treated DCs. Productions of both IL-10and TGF-b1 by PKCI-treated DCs were .4-fold and .2-foldhigher than those of mDCs on the fifth day after stimulation ofproinflammatory cytokines or LPS, respectively (Fig. 5A). Con-sequently, PKCI-treated DCs retained their T cell hypores-ponsiveness on the first day and the fifth day after stimulation (Fig.5B). There were no significant differences of production of bothIL-10 and TGF-b1 and T cell hyporesponsiveness among Bis I–,Go6983-, and Ro32-0432–treated DCs. In contrast, iDCs matured

and showed upregulated expression of costimulatory moleculessuch as CD80, CD83, and CD86, resulting in loss of T cell hy-poresposiveness upon addition of proinflammatory mediators.These findings indicated that PKCI-treated DCs are refractory to

FIGURE 3. PKCI-treated human DCs induce IL-10–producing T cells

and Foxp3+ regulatory T cells. (A) Comparison of IL-10–producing T cells

and Foxp3+ T cells induced by iDCs, mDCs, and PKCI-treated DCs. Naive

CD4+CD45RA+ T cells (53 106) were cultured with 53 105 MMC-treated

allogeneic iDCs, mDCs, or PKCI-treated DCs for 5 d. Then, the intracellular

expression of IL-10 and Foxp3 in CD4+ T cells and CD4+CD25+ T cells was

analyzed by flow cytometry. Numbers in the corners indicate the percentage

of positive cells. (B) CD4+ T cells cocultured with PKCI-treated DCs sup-

press the proliferation of responder T cells. Naive CD4+CD45RA+ T cells

(53 106) were cultured with 53 105 MMC-treated allogeneic iDCs, mDCs,

or PKCI-treated DCs for 5 d. Then, CD4+ T cells were purified from each

coculture. Purified CD4+ T cells (1 3 105) were incubated with syngeneic

Th1 cells (responder T cells, 1 3 105) in the presence of MMC-treated

allogeneic mDCs (1 3 104) for 4 d. The proliferation of responder T cells

was determined using [3H]thymidine incorporation. The proliferation of

responder T cells with mDCs (control) was 42,669 6 2,571 cpm. The

proliferation of responder T cells with mDCs by adding CD4+ T cells

cocultured with mDCs, iDCs, or PKCI-treated DCs was expressed relative to

that of control. Representative data from three experiments are shown. *p ,0.01 versus control. (C) Both direct cellular contact and soluble factors

mediate the inhibitory effect of CD4+ T cells cocultured with Bis I–treated

DCs. Coculture experiments were also performed without anti–IL-10 and

anti–TGF-b mAbs (no Abs-C) or in the presence of saturating blocking

anti–IL-10 (40 mg/ml) and/or anti-TGF–b1 (40 mg/ml) mAbs. The addition

of isotype control (IgG) did not alter the effect of CD4+ T cells cocultured

with Bis I–treated DCs on the proliferation of responder T cells. Transwell

experiments were performed using 24-well plates. Responder T cells (5 3105) together with MMC-treated allogeneic mDCs (53 104) were placed in

the bottom wells. CD4+ T cells (53 105) generated in the presence of Bis I–

treated DCs were placed in the upper wells. The cultures were performed no

Abs-C or in the presence of anti–IL-10 and/or anti–TGF-b1 mAbs for 4 d.

Then, the basket was removed and the proliferation of the responder T cells

was measured using [3H]thymidine incorporation. The control value in co-

culture experiment was 42,669 6 2,571 cpm. The control value in the

Transwell experiment was 25,940 6 1,786 cpm. The proliferation of re-

sponder T cells in each condition was expressed relative to that of control.

Representative data from three experiments are shown. *p , 0.05 versus no

Abs-C, **p , 0.01 versus no Abs-C, †p , 0.01 versus control (Transwell).

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stimulation with proinflammatory mediators and that their tol-erogenic properties are highly stable.

PKCI reduces maturation stimuli–induced increases in NF-kBfamily members in DCs

It has been reported that a crucial pathway for the maturation ofDCs upon exposure to proinflammatory stimuli involves the tran-scription factor NF-kB (57–60). iDCs pretreated with DMSO(control) or PKCIs were exposed to TNF-a, IL-1b, and/or PGE2

and the resulting effects on the nuclear NF-kB family members,RelA (p65) and RelB, were measured. As shown in Fig. 6, TNF-astimulation increased the expression of RelA and RelB 2.8- and2.2-fold, whereas pretreatment with PKCIs reduced their expres-sion 1.5- to 2.0- and 1.1- to 1.5-fold, respectively, relative to thatof unstimulated iDCs. Alternatively, expression of RelA and RelBwas increased 1.5- and 1.3-fold, respectively, upon stimulationwith IL-1b and declined to the baseline level upon treatment withPKCI. In contrast, expression of RelA and RelB did not increasesignificantly upon stimulation with PGE2. Upon stimulation witha maturation mixture containing TNF-a, IL-1b, and PGE2, ex-pression of RelA and RelB was increased synergistically 3.5- and3.0-fold and was reduced 1.5- to 1.8- and 1.3- to 1.5-fold by PKCItreatment, respectively. This also indicated that expression ofRelA and RelB in PKCI-treated DCs is slightly higher than that iniDCs. Additionally, these results demonstrate that conventionalPKC is required for both the canonical (RelA) and noncanonical

(RelB) pathways of NF-kB activation, and that conventional PKCplays a critical role in DC maturation.

PKCI increases intracellular cAMP in DCs upon stimulationwith a maturation mixture

Intracellular cAMP/CREB has been reported to play a critical rolein transcriptional regulation of IL-10 in monocytes and DCs (61,62). iDCs pretreated with DMSO (control) or PKCI were stimu-lated with TNF-a, IL-1b, and/or PGE2, and production of intra-cellular cAMP was analyzed. As shown in Fig. 7, upon stimulationwith TNF-a, production of cAMP was hardly recognizable buta small amount of cAMP was produced upon treatment withPKCI. IL-1b stimulation led to little or no production of cAMP,even with PKCI treatment. In contrast, cAMP production wasincreased significantly by PGE2 stimulation, but a low amount ofIL-10 was produced. Upon treatment with PKCI, production ofcAMP and IL-10 was increased 2-fold and .5-fold, respectively,

FIGURE 4. Phenotypic stability of PKCI-treated human DCs upon

proinflammatory stimulation. After removal of a maturation mixture and

PKCIs, iDCs, mDCs, or PKCI-treated DCs were cultured in the absence or

presence of a mixture of proinflammatory cytokines containing IFN-g

(1000 U/ml), IL-1b, IL-6, and TNF-a each at 10 ng/ml, or LPS (0.1 mg/ml).

After 1 and 5 d, DCs were analyzed for cell surface phenotype by flow

cytometry. Values are the means 6 SD of mean fluorescence intensity

(MFI) of each phenotype on DCs from four experiments (n = 2 or 3 sam-

ples/experiment). *p , 0.05 versus mDCs, †p , 0.01 versus mDCs.

FIGURE 5. Stability of PKCI-treated human DCs upon proinflammatory

stimulation. (A) Production of IL-10 and TGF-b of PKCI-treated DCs upon

proinflammatory stimulation. After removal of a maturation mixture and

PKCIs, iDCs, mDCs, or PKCI-treated DCs were cultured in RPMI 1640

medium supplemented with 10% FBS or in X-VIVO medium only for assay

of TGF-b in the absence or presence of a mixture of proinflammatory

cytokines or LPS. After 5 d, production of IL-10 and TGF-b in the culture

supernatants was determined by ELISA. Values are the means 6 SD of four

experiments (n = 2 or 3 samples/experiment). *p , 0.01. (B) Suppressive

properties of PKCI-treated DCs upon proinflammatory stimulation. DCs were

washed after stimulation with a mixture of proinflammatory cytokines or LPS

and recultured in RPMI 1640 medium supplemented with 10% FBS for a

further 24 h. Then, an MLR was performed as described in the in vitro T cell

proliferation assay section. T cell proliferation by iDCs and PKCI-treated DCs

was expressed relative to that by mDCs. Values are the means 6 SD (n = 6/

sample). Representative data from four experiments are shown. *p , 0.05

versus mDCs, **p , 0.01 versus mDCs.

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relative to DCs stimulated with PGE2 alone. Upon stimulation witha maturation mixture, intracellular cAMP production was reducedsignificantly compared with that of DCs stimulated with PGE2

alone. However, upon treatment with PKCI, production of cAMPand IL-10 recovered to a level that was the same as, or higher than,that of DCs treated with PGE2 and PKCI. These findings indicatedthat PGE2 acts antagonistically to TNF-a or IL-1b in terms ofcAMP production by DCs, and that this antagonistic effect isstrongly attenuated by PKCI. Additionally, upregulation of intra-cellular cAMP in DCs does not, in itself, induce IL-10 production,and the presence of PKCI is additionally necessary for this effect.

Characterization of PKCI-treated mouse DCs

First, to examine the effect of PKCI-treated DCs in vivo in a murinemodel of acute GVHD, we tested whether it would be possible togenerate PKCI-treated mouse DCs that had similar properties toPKCI-treated human DCs using a maturation mixture (TNF-a, IL-1b, and PGE2). Mouse iDCs were generated by culturing BM cellswith GM-CSF for 6 d and then matured with LPS or a maturationmixture for a further 48 h. LPS was used as a maturation control.As shown in Fig. 8A, as well as LPS-induced mDCs, mDCs in-duced by the maturation mixture showed high expression levels ofCD40, CD80, CD83, CD86, MHC class I (H-2Kd), MHC class II(I-A/I-E), and CCR7. Functionally, mDCs induced by the matu-

ration mixture showed migration activity toward CCL19 (Fig. 8B),high IL-12 production (Fig. 9A), sufficient proliferation of allo-geneic T cells (Fig. 9B), and low phagocytic capacity (Fig. 9C),similar to LPS-induced mDCs.PKCI-treated DCs were generated by culturing iDCs with

a maturation mixture in the presence of Bis I for 48 h. Bis I–treatedDCs showed markedly and significantly decreased levels of ex-pression of CD40, CD80, CD83, CD86, and MHC class I, andslightly decreased levels of expression of MHC class II, in com-parison with mDCs induced by the maturation mixture (Fig. 8A).Bis I–treated DCs showed CCR7 expression and migration activitytoward CCL19, similar to mDCs (Fig. 8B). Bis I–treated DCs alsohad high production of IL-10 and TGF-b, T cell hyporesponsive-ness, and efficient phagocytic capacity, as was the case for humanPKCI-treated DCs (Fig. 9A–9C). Thus, using a maturation mix-ture, we were able to generate mouse PKCI-treated DCs that hadproperties similar to those of human PKCI-treated DCs.

PKCI-treated DCs prevent acute GVHD

Next, we examined the therapeutic effect of PKCI-treated DCs onacute GVHD following allogeneic BMT. We transplanted lethallyirradiated BALB/c recipients (H-2d) with either T cell–depletedBM cells or with T cell–depleted BM plus spleen T cells (GVHDinduction) from C57BL/6 (H-2b) mice. Two days after BMT, we

FIGURE 6. PKCI suppresses maturation stimuli–induced increases in

RelA and RelB in human DCs. iDCs (1 3 106/well) were preincubated

with PKCI or DMSO (control) for 2 h and then stimulated with TNF-a (10

ng/ml), IL-1b (10 ng/ml), and/or PGE2 (1 mg/ml). After 4 h of stimulation,

activated NF-kBs (RelA or RelB) in the nuclear extracts were measured by

oligonucleotide-based ELISA as described in Materials and Methods. The

sample values were normalized against total cell protein. The content of

RelA and RelB in DCs after stimulation was expressed relative to that of

unstimulated iDCs (iDCs alone). Values are the means 6 SD of three

experiments. *p , 0.05, **p , 0.01.

FIGURE 7. PKCI increases intracellular cAMP and IL-10 production in

human DCs upon stimulation with a maturation mixture. After iDCs (2 3105/well) were cultured in a 96-well plate for 12 h, the cells were incu-

bated with PKCIs or DMSO for 1 h. The cells were washed with KRH

buffer, KRH buffer containing forskolin (10 mM) with or without PKCI

was added, and then the cells were stimulated with TNF-a, IL-1b, and/or

PGE2 for 1 h. The cell lysate was used for measurement of cAMP. To

examine IL-10 production, iDCs (2 3 105/well) were cultured in a 96-well

plate for 12 h. After the cells were incubated with PKCIs for 1 h, the cells

were stimulated with TNF-a, IL-1b, and/or PGE2 for 48 h. The super-

natants were harvested and assayed. Values are the means 6 SD of three

experiments. *p , 0.05, **p , 0.01.

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injected iDCs, mDCs, and Bis I–treated DCs obtained from BALB/cmice. As shown in Fig. 10, mice given only T cell–depleted BMcells appeared healthy, and 100% of the animals survived for atleast 50 d. Mice that received BM cells plus spleen T cells de-veloped severe signs of GVHD, including weight loss, reducedmobility, hunched posture, diarrhea, and ruffled fur, and they diedwithin 30 d. There were no significant differences of GVHD le-thality among untreated, iDC-treated, and mDC-treated mice. Incontrast, administration of Bis I–treated DCs protected the micefrom lethal GVHD, and 80% of them survived for 50 d (p, 0.01).From these findings, we confirmed that PKCI-treated DCs weremore efficient than iDCs and mDCs for preventing GVHD in vivo.

DiscussionIn this study, we demonstrated that human tolerogenic DCs weregenerated by adding a conventional PKC inhibitor during thematuration process after iDC differentiation, but not during theprocess of differentiation from progenitors to iDCs. The PKCI-treated DCs had a semimature phenotype with high productionof IL-10, and they induced IL-10–producing T cells and functionalregulatory T cells from naive CD4+ T cells efficiently, thus elicitinga strong immunosuppressive function. These cells expressed CCR7

and had sufficient capacity to migrate toward CCR7 ligands, es-pecially secondary lymphoid organs. Additionally, PKCI-treatedDCs were highly stable upon exposure to inflammatory stimuli.Moreover, PKCI-treated mouse DCs that had properties similar toPKCI-treated human DCs prevented acute GVHD in a murinemodel.DCs are a heterogeneous group of APCs involved in the initiation

of both adaptive immunity and tolerance induction. Generally,tDCs are characterized by an immature or semimature phenotype,with low expression of costimulatory molecules, low production ofproinflammatory cytokines, including IL-12 and TNF-a, and highproduction of anti-inflammatory cytokines such as IL-10 and TGF-b in comparison with mDCs (2–6). Partial maturation resulting in

FIGURE 8. PKCI suppresses expression of costimulatory molecules on

mouse DCs. (A) Surface phenotype on iDCs, mDCs, and PKCI-treated DCs.

BM cells (2 3 106) obtained from BALB/c mice were incubated in RPMI

1640 medium with 10% FBS and 20 ng/ml rmGM-CSF for 6 d. Then,

nonadherent cells (iDCs) were collected and stimulated with LPS (1 mg/ml)

or a maturation mixture containing rmTNF-a (50 ng/ml), rmIL-1b (10 ng/

ml), and PGE2 (200 ng/ml) for 48 h to induce maturation. PKCI-treated

DCs were generated by culturing iDCs with a maturation mixture in the

presence of Bis I (10 mM) for 48 h. Surface phenotype on DCs was analyzed

by flow cytometry. Numbers in the corners indicate the mean fluorescence

intensity (MFI) of expression level of each surface marker. Representative data

from three experiments are shown. (B) Chemotactic activity of iDCs, mDCs,

and PKCI-treated DCs to CCL19. Aliquots (100 ml) of cells (5 3 106/ml)

were added to the upper chambers, and CCL19 (1 mg/ml) was added to the

lower wells. After 2 h, cell migration was quantified by counting cells in each

lower chamber and cells adhering to the bottom part of the filter. Each assay

was performed in triplicate. *p , 0.01.

FIGURE 9. Characterization of mouse iDCs, mDCs, and PKCI-treated

DCs. (A) Cytokine production from iDCs, mDCs, and PKCI-treated DCs.

iDCs, mDCs, and Bis I–treated DCs from BALB/c mice were prepared as

described inMaterials and Methods. These DCs (13 106/ml) were cultured

in RPMI 1640 medium supplemented with 10% FBS for assay of IL-6, IL-

10, IL-12p70, and IFN-g, or in X-VIVO medium only for assay of TGF-b.

The cell supernatants were harvested after 96 h and assayed. Values are the

means 6 SD of three experiments. *p , 0.01. (B) Comparison of sup-

pressive properties among iDCs, mDCs, and PKCI-treated DCs. mouse

CD4+ T cells isolated from spleens of C57BL/6 mice (1 3 105) were

cocultured with 1 3 104 (10:1 T cell/DC ratio) or 2 3 104 (10:2) MMC-

treated allogeneic (BALB/c mice) iDCs, mDCs, and Bis I–treated DCs.

After 4 d in culture, T cell proliferation was determined using [3H]thymi-

dine incorporation. T cell proliferation by iDCs and Bis I–treated DCs was

expressed relative to that by mDCs induced by a maturation mixture. Values

are the means 6 SD (n = 6/experiment). Representative data from three

experiments are shown. *p , 0.05, **p , 0.01. (C) Phagocytic ability of

iDCs, mDCs, and PKCI-treated DCs. DCs were incubated with FITC/latex

beads/rabbit IgG for 24 h and then analyzed by flow cytometry.

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intermediate expression of MHC and costimulatory molecules,but little or no production of proinflammatory cytokines, has beentermed semimaturation (2). The mechanisms by which tDCs in-duce tolerance include 1) deletion of T cells, 2) induction of T cellhyporesponsiveness, 3) deviation of the T cell cytokine profile,and 4) induction of regulatory T cells (7). The anti-inflammatorycytokines IL-10 and TGF-b, neuropeptides including vasoactiveintestinal peptide and a-melanocyte–stimulating hormone, andseveral pharmacological agents such as Dexa, Rapa, and Vit D3

have been reported to promote or restore Ag-specific tolerance inexperimental animal models (8–17, 21, 27, 29, 31, 35).On the basis of these data, clinical trials of ex vivo–generated

tDCs are currently being considered. Three functional character-istics are required for clinically applicable tDCs (37–39). First, exvivo–generated tDCs need to have the capacity for CCR7-dependent migration toward secondary lymphoid organs for in-duction of suppressive responses in naive T cells. Second, in theseorgans, tDCs should be capable of inducing the right type ofimmunosuppressive effector mechanism, especially efficient in-duction of functional regulatory T cells. Third, it is essential toestablish whether ex vivo–generated tDCs are stable and maintaintheir tolerogenic properties when encountering proinflammatorysignals in vivo. Two groups have compared migratory capacity,stability, and functional properties among clinical-grade tDCsgenerated with Vit D3, Dexa, Rapa, IL-10, or TGF-b (38, 39). VitD3-tDCs, Dexa-tDCs, and IL-10-tDCs had very low CCR7 ex-pression, leading to very poor migration to CCL19 and CCL21,whereas TGF-b-tDCs and Rapa-tDCs had moderate and highCCR7 expression, respectively. Vit D3-tDCs, Dexa-tDCs, IL-10-tDCs, and TGF-b-tDCs showed an immature or semimaturephenotype, whereas the phenotype of Rapa-tDCs resembled thatof mDCs. IL-10 production by Vit D3-tDCs, Dexa-tDCs, and IL-10-tDCs was moderate, whereas that of TGF-b-tDCs and Rapa-

tDCs was slight or undetectable. Functionally, IL-10-tDCs and VitD3-tDCs suppressed T cell activation, whereas TGF-b-tDCshardly did so. Two groups have obtained conflicting data regard-ing the suppressive activity of Dexa-tDCs and Rapa-tDCs. All fivetDCs maintained stable tolerogenic properties under proinflammatoryconditions. Overall, IL-10-tDCs appear to possess the most pow-erful tolerogenic characteristics with high IL-10 production and arethe best inducers of regulatory T cells, but they show the poorestmigration toward CCR7 ligands. In the present study, PKCI-treatedDCs showed moderate expression of CCR7 and had sufficientcapacity to migrate toward CCR7 ligands. PKCI-treated DCsstrongly suppressed T cell activation by generating IL-10–pro-ducing T cells and functional Foxp3+ regulatory T cells from naiveCD4+ T cells. Additionally, their tolerogenicity was highly stablein the presence of proinflammatory stimuli. PKCI-treated humanDCs prepared in serum-free X-VIVO 15 medium had propertiessimilar to those of cells prepared in RPMI 1640 medium with 10%FBS. From these findings, PKCI-treated human DCs satisfy theabove three requirements for clinical application. Moreover, wesuccessfully generated PKCI-treated mouse DCs that had similarproperties to PKCI-treated human DCs and demonstrated thatPKCI-treated DCs prevented acute GVHD in vivo. Taken together,these findings suggest that PKCI-treated DCs could be clinical-grade tDCs.Members of the PKC family are serine-threonine protein kinases

that are divided into three subfamilies, conventional, novel, andatypical PKCs, depending on their responsiveness to calcium andDAG stimulation (40). PKCs are activated by PMA, an activator ofconventional PKC-derived progenitors such as CD34+ hemopoieticprogenitors and CD14+ monocytes, to undergo DC differentiation(41, 44, 45). GM-CSF, IL-4, and TNF-a each activates PKC orPKCb signaling, and addition of these cytokines leads to differen-tiation of progenitors to DCs (63–65). Other groups have reportedthat PKCbI and PKCbII play an essential role in DC differentiationand that PKCa signaling is a key driver of monocyte-to-macrophagedifferentiation (44, 45). Our present study showed that stable, tol-erogenic DCs with a semimature phenotype were generated byaddition of conventional PKCI during the maturation process. Toexamine which of the conventional PKCIs affected DC toler-ogenicity, we compared the downregulation of costimulatory mol-ecules (CD40, CD80, and CD86) using the specific PKCIs Go6976(a PKC a- and bI-specific inhibitor), CGP53353 (a PKC bII-specificinhibitor), and HBDDE (a PKC a- and g-specific inhibitor). Bis I–,Go6976-, CGP53353-, and HBDDE-treated DCs all showed down-regulation of costimulatory molecules. Of these, Bis I–treated DCsshowed the strongest downregulation, indicating that PKC a, b,and g are all involved in the expression of costimulatory moleculeson DCs (Supplemental Fig. 4). Taken together, these findingssuggest that the conventional PKC subfamily plays a critical rolenot only in DC differentiation but also maturation in response toexogenous stimuli.Studies involving culture with cytokines including GM-CSF,

IL-4, and TNF-a have indicated that PKCbΙΙ plays an essential rolein DC differentiation through noncanonical/RelB-mediated NF-kBsignaling (44). PKCε is involved in LPS-induced IL-12 productionby DCs through IkB-a degradation and subsequent NF-kB acti-vation (42). These findings suggest that NF-kB signaling may beinvolved in the mechanism by which conventional PKCI inducestDCs. NF-kB is required for proper development and function ofDCs, and its activation is upregulated early during differentia-tion from precursors such as CD34+ hemopoietic progenitors andmonocytes. Additionally, when matured in the presence of TNF-aalone or LPS, NF-kB activation plays an essential role in IL-12production and expression of MHC class II and costimulatory

FIGURE 10. PKCI-treated DCs prevent acute GVHD. Lethally irradi-

ated BALB/c recipients (H-2d) were transplanted with either T cell–de-

pleted BM cells (d) or with T cell–depleted BM plus spleen T cells (GVHD

induction) from C57BL/6 (H-2b) mice. Recipients received a single i.v.

injection of medium (s), host-matched iDCs (n), mDCs (▴), or Bis I–treated DCs (N) 2 d after GVHD induction. Recipients were monitored

daily from the day of transplantation until they died naturally of GVHD.

Differences in survival of treatment groups in the GVHD experiment

were analyzed using Kaplan–Meier and log-rank test. *p , 0.01 versus

any other groups. Similar results were obtained from two independent

experiments.

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molecules such as CD80, CD86, and CD40 in DCs (58). Whenmatured with a mixture of TNF-a, IL-1b, and PGE2, DCs treatedwith NF-kB inhibitors BAY 11-7082 or aspirin have been shownto express high levels of MHC class II and CD86 but markedlyreduced expression of CD40 and generated anergy and regulatoryT cell activity (66). NF-kB family members include RelA (p65),RelB, p52/p100, p50/p105, and c-Rel. The heterodimers RelA/p50and RelB/p52 are required for activation of the canonical andnoncanonical pathways, respectively. Inhibitory IkBs can be de-graded by the proteasome to allow nuclear translocation of NF-kBand activate transcription in the canonical pathway (60). Amongthe NF-kB family members, RelB is the most crucial NF-kBsubunit for expression of CD40, CD86, and MHC class II on DCsduring maturation (57, 67, 68). RelB-deficient mice show impair-ment of APC function and cellular immunity (67). Ag-primed DCswith inhibited RelB function have been shown to lack typical co-stimulatory molecules (68). In the present study, DC maturationinduced by TNF-a or IL-1b was NF-kB–dependent, whereas thatinduced by PGE2 was NF-kB–independent. Moreover, conven-tional PKCIs inhibited NF-kB activation of both the canonical andnoncanonical pathways of DC maturation, leading to suppressionof costimulatory molecule expression and IL-12 production andresulting in a semimature state.The cAMP/CREB-dependent pathway plays a critical role in IL-

10 production by DCs (61, 62). However, an increase of the in-tracellular level of cAMP by PGE2 reportedly has its own limitedeffect on IL-10 production (62), and this is reflected in our presentdata. Additional stimulation is necessary to elicit a synergisticincrease of IL-10 production with cAMP. Competition betweenNF-kB and cAMP/CREB for IL-10 gene transcription has beenreported (69, 70), and NF-kB inhibition is associated with an in-crease of IL-10 production (62, 71, 72). In the present study, uponstimulation with PGE2 alone or a maturation mixture includingTNF-a, IL-1b, and PGE2, the amount of intracellular cAMP inPKCI-treated DCs increased only 2- to 3-fold relative to that inPKCI-untreated DCs. However, IL-10 production in PKCI-treatedDCs showed a much greater increase (.5-fold) than that in PKCI-untreated DCs. This augmentation of IL-10 production may bea synergistic effect of increased cAMP and NF-kB inhibitionelicited by conventional PKCIs.tDCs are a promising therapeutic tool for specific cellular in-

duction of immunological tolerance in the context of transplantationand autoimmunity. As there will be a demand for clinical-gradetDCs for tolerance-inducing therapy, PKCI-treated tDCs may besuited for this purpose.

DisclosuresThe authors have no financial conflicts of interest.

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