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TISSUE-SPECIFICSTEMCELLS

Periodontal Ligament Stem Cells Regulate B Lymphocyte FunctionVia Programmed Cell Death Protein 1

OUSHENG LIU,a,b JUNJI XU,

a GANGDING,a DAYONG LIU,

a ZHIPENGFAN,a CHUNMEI ZHANG,

a WANJUN CHEN,c

YAOZHONG DING,d ZHANGUI TANG,b SONGLINWANGa,e

aSalivary Gland Disease Center and Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction,

Capital Medical University School of Stomatology, Beijing, China, bDepartment of Oral and Maxillofacial

Surgery, Xiangya Hospital, Central South University, Changsha, China, cNational Institute of Dental and

Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA, dDepartment of Immunology,

Capital Medical University, Beijing, China, eDepartment of Biochemistry and Molecular Biology, Capital Medical

University School of Basic Medical Sciences, Beijing, China

Key Words. Immunoregulation Mesenchymal stem cells Periodontal ligament stem cells Periodontitis B lymphocyte Humoral

immunity Programmed cell death protein 1

ABSTRACT

Periodontal ligament stem cells (PDLSCs) have providednovel cell sources for tooth and periodontal tissue regener-ation. Allogeneic PDLSCs can reconstruct periodontal liga-ment tissue that has been damaged by periodontal diseasesand regulate T-cell immunity. However, the effect ofPDLSCs on B cells remains unknown. Here, we treatedperiodontitis in a miniature pig model using allogeneicPDLSCs and showed a reduction in humoral immunity inthe animals. When cocultured with normal B cells, humanPDLSCs (hPDLSCs) had similar effects as bone marrow

mesenchymal stem cells in suppressing B cell proliferation,differentiation, and migration, while intriguingly, hPDLSCsincreased B cell viability by secreting interleukin-6. Mecha-nistically, hPDLSCs suppressed B cell activation throughcell-to-cell contact mostly mediated by programmed celldeath protein 1 and programmed cell death 1 ligand1. Our data revealed a previously unrecognized function ofPDLSCs in regulating humoral immune responses, whichmay represent a novel therapeutic strategy for immune-related disorders. STEM CELLS 2013;31:13711382

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Adult stem cells exist in many tissues, including bone mar-row, skin, adipose tissue, tendon, lung, heart, liver, placenta,and umbilical cord blood [19]. Due to their low immunoge-nicity and immunoregulatory function, mesenchymal stemcells (MSCs) play a key role in tissue regeneration and havebeen used in clinical trials in therapy for severe refractory dis-eases [1012].

Periodontitis is one of the most widespread chronic infec-tious diseases in humans, which is the most common cause fortooth loss in adults. Several factors are known to be involvedin the occurrence of periodontitis [13, 14]. Our previous studyshowed that periodontal ligament stem cells (PDLSCs) from

periodontitis tissue had impaired immunomodulatory function,which may lead to an imbalanced immune response and theacceleration of osteoclastogenesis and inflammation relatedbone loss [15]. PDLSCs and other tooth-related stem cells haveprovided new prospects and potential therapeutic cell sourcesfor tooth regeneration and the reconstruction of periodontal

ligament tissue damaged by periodontal diseases [1618]. How-ever, limited cell sources of autologous dental stem cell popu-lations impede the potential of clinical application.

Allogeneic dental stem cells significantly enlarged thesource of seed cells for teeth and periodontal tissue regenera-tion and reconstruction development. We demonstrated previ-ously that allogeneic PDLSCs exhibited immunosuppressiveactivities on activated T-cells in vitro [18]. In vivo allogeneicPDLSCs can regenerate and reconstruct periodontal ligamentdamaged by periodontal diseases and inactivate T-cell immu-nity. However, the effects of PDLSCs on B cells areunknown. Here, we investigated whether allogeneic PDLSCscould affect B-cell immune responses in vitro and in vivo.We also studied human PDLSC (hPDLSC)-mediated regula-tion of human B cells. We showed that allogeneic PDLSCs

failed to activate humoral immunity in vivo in miniaturepigs. We also demonstrated that hPDLSCs inhibit human Bcell proliferation, differentiation, and chemotactic behavior.hPDLSCs secreted interleukin (IL)-6 and enhanced B-cell sur-vival. The immunoregulatory function of hPDLSCs in humanB cells was achieved by programmed death-1 (PD-1) and its

Author contributions: O.L., J.X., and S.W.: designed the study and wrote the manuscript; O.L., J.X., G.D., D.L., and S.W.: performedresearch and analyzed the data; Z.F.; C.Z., Y.D., and Z.T.: analyzed the data and technical support; W.C.: provided critical input on theimmunological studies; S.W.: got funding and approved the manuscript. O.L. and J.X. contributed equally to this article.

Correspondence: Songlin Wang, D.D.S., Ph.D., Capital Medical University School of Stomatology, Tian Tan Xi Li No. 4, Beijing100050, China. Telephone: 86-10-83911708; Fax: 86-10-67062012; e-mail: [email protected] Received October 24, 2012; RevisedFebruary 19, 2013; accepted for publication March 7, 2013; first published online in STEM CELLS EXPRESS April 3, 2013; available onlinewithout subscription thorugh the open access option. VC AlphaMed Press 1066-5099/2013/$30.00/0 doi: 10.1002/stem.01387

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ligand (PD-L1) interaction in cell-to-cell contact dependentmanner.

MATERIALS ANDMETHODS

PDLSC-Mediated Treatment for Swine Periodontitis

Bama inbred miniature pigs (68 months old, weighing 1019kg) were obtained from the Institute of Animal Science of theDong Bei Agriculture University, China, http://www.neau.edu.cn/english. Miniature pigs were kept under conventionalconditions with free access to water and a regular supply ofsoft food. We generated periodontitis lesions in 12 femaleminiature pigs as reported previously [17], for a total of 24defects. For the treatment study, defects were randomlyassigned to four groups: control, hydroxyapatite/tricalciumphosphate (HA/TCP, 40 mg) alone, HA/TCP scaffolds plusautologous pPDLSCs (4.0 106 cells), and HA/TCP scaf-folds plus allogeneic pig PDLSCs (pPDLSCs, 4.0 106

cells). All protocols for swine were approved by the Animal

Care and Use Committee of Capital Medical University.Details are described in Supporting Information Methods.

Clinical Assessment of Periodontal TissueRegeneration

Clinical assessments, including probing depth, gingival reces-sion, and attachment loss, were measured on all experimentalteeth before the generation of the periodontitis models (week4), before the treatment (week 0), and 12 weeks post-trans-plantation. Routine blood tests (white blood cells, red bloodcells, platelets, and hemoglobin concentration), routine biochem-ical tests (aspartate aminotransferase, alanine aminotransferase,total protein, blood urea nitrogen, and creatinine), and immuno-globulins (IgG, IgA, and IgM) in blood and gingival crevicularfluid were examined at week 4; at 1, 3, 5, and 7 days post-

transplantation; and at 2, 4, 8, and 12 weeks post-transplanta-tion. B cell-related markers (percentage of CD20 cells, CD25

cells, and B220 cells) were examined at 3 and 7 days post-transplantation and at 2, 4, 8, and 12 weeks post-transplantation.A calibrated volumetric micropipette of 5 lL capacity wasintroduced into the periodontal pocket of the selected site forcollection of gingival crevicular fluid by Brill technique. Thesample was collected for 20 minutes. The collected sample wasthen transferred to a sterilized plastic vial with the help of air-spray. The samples of GCF were stored in plastic vials at70C until analyzed for antibodies level [19, 20].

Periodontal tissues were taken from autologous and allo-geneic groups. In these regenerated tissues, IgG, IgA, andIgM were examined by enzyme-linked immunosorbent assay(ELISA) at 3 days and 7 days post-transplantation. In addi-

tion, real-time polymerase chain reaction assays for IL-6, PD-1, PD-L1, and programmed cell death 1 ligand 2 (PD-L2)were evaluated at 1 and 2 weeks post-transplantation, asdescribed in Supporting Information Methods.

Imaging and Histological Assessments

Bone regeneration was examined by computed tomography(Siemens, Germany, http://www.usa.siemens.com/answers/en)scanning on transplantation (week 0) and at 12 weeks post-transplantation. At 12 weeks post-transplantation, all animalswere killed; samples from the experimental areas were har-vested, fixed with 4% formaldehyde, decalcified with 50%formic acid, and embedded in paraffin. Sections (5 lm) weredeparaffinized and stained with hematoxylin and eosin. Forimmunohistological costaining for PDLSCs and B cells, anti-

CD146 (Abcam, U.K., http://www.abcam.com) and anti-CD19(R&D, Minneapolis, MN, US, http://www.rndsystems.com)antibodies were used.

Flow Cytometry

hPDLSCs were harvested, and aliquots (1.0 106 cells) were

incubated for 40 minutes with specific monoclonal antibodiesagainst STRO-1 (R&D, Minneapolis, MN, US, http://www.rndsystems.com), CD146 (Abcam, U.K., http://www.abcam.com), CD90 (Abcam, U.K., http://www.abcam.com),CD73 (BD, Biosciences, San Jose, CA, US, http:// www.bdbiosciences.com), or SSEA-4 (R&D, Minneapolis,MN, US, http://www.rndsystems.com). Purified B cells werecocultured without or with hPDLSC suspensions at the indi-cated ratio in the presence of CpG oligodeoxynucleotide(ODN), rCD40L, anti-immunoglobulin (anti-Ig), IL-2, and IL-4 for 72 hours. Human bone mesenchymal stem cells(hBMSCs) were used as a positive control. For intracellularstaining experiments, B cells were cocultured for 6 hours withor without hPDLSC suspensions at the indicated ratios in thepresence of CpG ODN, rCD40L, anti-Ig, IL-2, IL-4, ionomy-cin (250 ng/ml), and GolgiPlug protein transport inhibitor(1 ll/ml). See details in Supporting Information Methods.

Human and Miniature Pig PDLSC and BMSCCultures

All protocols for the handling of human tissue were approvedby the Research Ethical Committee of Capital Medical Univer-sity, China. Isolation and culture of hPDLSCs and miniaturepig PDLSCs (pPDLSCs) are performed as reported previously[16, 17]. All cells used in this study were at 34 passages. Foreach experiment, we used hBMSCs (purchased from AmericanType Culture Collection, Manassas, VA, US, http://www.atcc.org) and pBMSCs at the same number of passages.

Differentiation of Multipotent PDLSCs

Multilineage differentiation assays toward osteogenic and adi-

pogenic pathways were performed as reported previously [21,22]. To detect osteogenic differentiation, calcification of theextracellular matrix was checked on day 5 by alkaline phos-phatase (ALP) staining and on day 14 by von Kossa staining.Oil red O staining was used to identify lipid-laden fat cells.

B Lymphocyte Isolation and Culture

Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient (Sigma, St. Louis, MO, US, http://www.sigmaaldrich.com) from the peripheral blood of sixhealthy donors. Briefly, cell suspensions were indirectly mag-netically labeled using a cocktail of biotin-conjugated antibod-ies against CD2, CD14, CD16, CD36, CD43, and CD235awith anti-biotin microbeads (Miltenyi Biotech, Germany,http://www.miltenyibiotec.com). Negatively selected cells

were assessed by flow cytometric analysis with a CD19monoclonal antibody. All cell cultures were performed inRPMI 1,640 medium supplemented with 15% fetal bovine se-rum (FBS), 100 U/ml penicillin, and 100 lg/ml streptomycin.Cultures were incubated at 37C in a 5% CO2 atmosphere, inthe absence or presence of the following stimuli: the CpG syn-thetic oligonucleotide (2.5lg/ml; Oligonucleotide, Sangon Bio-tech, Shanghai, China, http://www.san gon.com), recombinantCD40L (rCD40L; 100 ng/ml; R&D, Minneapolis, MN, US,http://www.rndsystems.com), goat anti-human immunoglobulinantibodies (2 lg/ml; Immunotech, Jackson, West Grove, PA,USA, http://www.jacksonimmuno.com/home/disteuro.asp), IL-2(50 U/ml; R&D, Minneapolis, MN, US, http://www.rndsystems.com), IL-4 (10 ng/ml, R&D), and IL-10 (10 ng/ml;R&D, Minneapolis, MN, US, http://www.rndsystems.com).

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Proliferation Assay

Purified B cells were labeled with 2 lM carboxyfluoresceinsuccinimidyl ester (CFSE; Invitrogen, Carlsbad, CA, US,http://www.invitrogen.com). Then, B cells were cocultured inflat-bottomed 6-well plates (Corning, Costar, MA, US, http://www.corning.com/lifesciences) with hPDLSC suspensions from

allogeneic donors at ratios of 5:1, 1:1, and 1:5 in the presenceof CpG ODN, rCD40L, anti-Ig, IL-2, and IL-4 in a total vol-ume of 3 ml RPMI 1,640 medium per well in triplicate.hBMSCs were used as a positive control. After 3 days, B cellswere collected and analyzed using flow cytometry (BD Bio-sciences, San Jose, CA, US, http://www.bdbiosciences.com). Inanother assay, B cells labeled with CFSE were centrifuged,resuspended in supernatants from hPDLSCs or hBMSCs grownto confluence, and cultured for 3 days as described.

Apoptosis Assay

The percentage of apoptotic B cells after1 and 3 days of cul-ture with or without hPDLSCs or hBMSCs, in the presence ofCpG ODN, rCD40L, anti-Ig, IL-2, or IL-4, was evaluatedusing the FITC Annexin V Apoptosis Detection kit I (BD

Pharmingen, San Jose, CA, US, http://www.bdbiosciences.com) according to the manufacturers instructions.

Transwell Cultures

Transwell chambers with a 0.4-lm pore size membrane (Corn-ing, Costar, MA, US, http://www.corning.com/lifesciences) wereused to physically separate the B lymphocytes from thehPDLSCs. Purified B cells (2.0 105 cells) were labeled with2 lM CFSE and seeded in the upper chamber, and hPDLSCs(2.0 105 cells) were placed in the bottom chamber, in thepresence of CpG ODN, rCD40L, anti-Ig, IL-2, and IL-4 in atotal volume of 2 ml RPMI 1,640 medium per well in triplicate.hBMSCs were used as a positive control. After 3 days, B cellswere collected and analyzed using flow cytometry (BD Bio-sciences, San Jose, CA, US, http://www.bdbiosciences.com).

Migration Assay

B-cell chemotaxis was investigated using 24-transwell plates witha 4-lm pore-size polycarbonate membrane (Corning, Costar, MA,US, http://www.corning.com/lifesciences). The migration assaywas performed as reported previously [23]. Briefly, purified Bcells were cocultured for 24 hours without or with hPDLSC sus-pensions at a ratio of 1:1 in the presence of CpG ODN, rCD40L,anti-Ig, IL-2, and IL-4. hBMSCs were used as a positive control.Next, 2.0 105 B cells were dispensed in the upper transwellchamber, while chemokines or medium alone were added to thelower chamber. CXCL12, CXCL13, and CCL19 were tested at300 ng/ml, which is a concentration chosen on the grounds ofdoseresponse experiments performed with normal tonsil B cells[23, 24]. Plates were incubated for 2 hours at 37C. Cells that

migrated into the lower chambers were harvested, stained withCD19 mAb, counted, and calculated as the percent ratio betweenthe number of B cells dispensed in the upper chamber and thenumber of B cells recovered from the lower chamber after migra-tion. The difference between input obtained following chemokinestimulation and that obtained after incubation with medium alonewas statistically analyzed as net percent input.

ELISA and Cytometric Bead Array (CBA) forAntibody Detection

Purified B cells were cocultured using 12-transwell plates in atotal volume of 3 ml RPMI 1,640 medium per well in tripli-cate, without or with hPDLSC suspensions at the indicatedratios in the presence of CpG ODN, rCD40L, anti-Ig, IL-2,IL-4, and IL-10. hBMSCs were used as a positive control.

After 1 week, supernatants were collected and tested byELISA and CBA assay (BD Biosciences, San Jose, CA, US,http://www.bdbiosciences.com). Details are described inSupporting Information Methods.

Measurement of Soluble Factors and Cytokines in

SuspensionPurified B cells alone, hPDLSCs or hBMSCs cocultured withB cells, or hPDLSCs or hBMSCs alone were cultured in thepresence of CpG ODN, rCD40L, anti-Ig, IL-2, or IL-4. After1, 2, and 3 days, the respective supernatants were collected.ELISAs were used to quantify prostaglandin E2 (PGE2), andCBA assays were used to detect IL-2, IL-4, IL-6, IL-10, IL-12p70, tumor necrosis factor alpha (TNFa), interferon gamma(IFNc), and tumor growth factor beta (TGFb) with a humanTh1/Th2 cytokine kit, human IL-12p70 flex set, and humanTGF-b kit (BD Biosciences, San Jose, CA, US, http://www.bdbiosciences.com) according to the manufacturersinstructions.

IL-6 for Apoptosis and Proliferation

Purified B cells (2 105

cells/well) were cocultured with orwithout hPDLSCs or hBMSCs and with or without CpG ODN,rCD40L, anti-Ig, IL-2, or IL-4by adding purified NA/LE ratanti-human IL-6 antibody (BD PharMingen, San Jose, CA, US,http://www.bdbiosciences.com) to the cultures at concentrationsof 0, 50, 100, or 200 ng/ml. After 3 days, B cells were col-lected and the percentage of apoptotic B cells was evaluatedusing the FITC Annexin V Apoptosis Detection kit I accordingto the manufacturers instructions. For proliferation assay,CFSE-labeled B cells were collected after 3 days of cultureand counted using flow cytometry.

Mechanisms of hPDLSC- and hBMSC-MediatedImmunosuppression

hPDLSCs or hBMSCs (2 105 cells/well) were cocultured

with or without purified B cells at a ratio of 1:1 in the presenceof CpG, ODN, rCD40L, anti-Ig, IL-2, and IL-4. After 3 days,hPDLSCs or hBMSCs were collected and subjected to cyto-fluorometric analysis using monoclonal antibodies against PD-1, PD-L1, and PD-L2 (eBioscience, San Diego, CA, US, http://www.ebioscience.com). Blocking experiments were performedby adding anti-PD-1, anti-PD-L1, or anti-PD-L2 monoclonalantibodies (eBioscience, San Diego, CA, US, http://www.ebioscience.com) at concentrations of 0, 0.12, 0.25, or 0.5 lg/ml; or anti-PD-L1 and anti-PD-L2 monoclonal antibodies(eBioscience, San Diego, CA, US, http://www.ebioscience.com)at concentrations of 0.5 lg/ml to cultures in which purified Bcells had been labeled with 2 lM CFSE. After 3 days, B cellswere collected and analyzed with flow cytometry (BD Bio-sciences, San Jose, CA, US, http://www.bdbiosciences.com).

Statistical Analysis

Statistical significance was assessed using a two-tailed Stu-dents t test or analysis of variance; p < .05 was consideredstatistically significant.

RESULTS

Allogeneic pPDLSC Transplantation SuppressesPeriodontitis and Humoral Immune Responses inMiniature Pig

At 12 weeks after transplantation, periodontal tissue was onlypartially regenerated in the control (Fig. 1A, 1E, 1I, 1M) and

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Figure 1. (Continued)



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HA/TCP groups (Fig. 1B, 1F, 1J, 1P). Both autologous andallogeneic pPDLSC treatments significantly improved periodon-tal tissue regeneration compared with the HA/TCP group andcontrol group (Fig. 1). The autologous and allogeneic pPDLSCgroups did not differ significantly regarding probing depth,attachment loss, or gingival recession (Fig. 1Yiiii). To test the

immunological response induced by allogeneic pPDLSC trans-plantation, we analyzed B cell-associated markers (Fig. 2A2D),immunoglobulins (Fig. 2E2G), and routine blood and biochemi-cal tests in whole blood (Supporting Information Table S1AS1B), as well as immunoglobulins in gingival crevicular fluids(Fig. 2H2J). Allogeneic and autologous PDLSC-treated groupsdid not differ significantly regarding the aforementioned parame-ters. The immunoglobulin levels in regenerated periodontal tis-sues (Fig. 2K, 2L) did not differ significantly between allogeneicand autologous PDLSC-treated pigs. The data suggest therewere no immunological rejections in the animals that receivedallogeneic pPDLSC transplantation.

Effects of hPDLSCs on B-Cell Proliferation

A cell population from the periodontal ligament of the human

third molar was isolated. These cells were positive for MSCmarkers, including STRO-1, CD146, CD90, SSEA-4, OCT-4,and CD73, thus identifying them as hPDLSCs (Supporting In-formation Fig. 1A) [22]. After osteogenesis-induced culturingfor 5 and 14 days, hPDLSCs were detected by ALP stainingand vonKossa staining, respectively (Supporting InformationFig. 1B). hPDLSCs also possessed the potential to developinto oil red O-positive lipid-laden fat cells after 4 weeks ofculture with an adipogenesis-inducing medium (SupportingInformation Fig. 1B).

Positively selected B cell populations contained, on aver-age, 93.28% B cells, as assessed by flow cytometric analysiswith a CD19 monoclonal antibody (Supporting InformationFig. 1C). To examine whether hPDLSCs affect the B-cell pro-liferation, the hPDLSCs were cocultured with blood B cells

(93.3% CD19, Supporting Information Fig. 1C) prelabeledwith CFSE and stimulated with the CpG ODN, rCD40L, anti-Ig, IL-2, and IL-4. hPDLSCs inhibited B-cell proliferation ina cell-number-dependent manner (Fig. 3A, 3B). At the highestratio of B cells to hPDLSCs (1:5), hPDLSCs almost com-pletely suppressed B-cell proliferation. As expected, hBMSCsalso inhibited B-cell proliferation (Fig. 3A, 3B).

To investigate the potential involvement of apoptosis inhPDLSC- or hBMSC-mediated inhibition of B-cell prolifera-tion [18, 25], activated B cells that had been cocultured with-out or with hPDLSCs or hBMSCs at a 1:1 ratio for 1 and3 days were stained with Annexin V and propidium iodide.Frequencies of Annexin V apoptotic B cells detected in cul-tures performed with hPDLSCs or hBMSCs were less than

those without hPDLSCs or hBMSCs (Fig. 3D, 3E). Moreover,a decrease in the B cell number in periodontal tissues afterPDLSC treatment was observed (Supporting Information Fig.2). These findings indicate that hPDLSC-mediated inhibitionof B-cell proliferation was not caused by B cell death.

hPDLSCs Inhibit B-Cell Proliferation ThroughCell-to-Cell Contact and Soluble Factors

CFSE-labeled B cells were plated in the lower chamber of atranswell system, physically separated from hPDLSCs orhBMSCs (at a 1:1 ratio, in the upper chamber). For cellcellcontact experiments, B cells were cocultured with hPDLSCsor hBMSCs at a 1:1 ratio. We showed that hPDLSCs, similarto hBMSCs, inhibited B-cell proliferation under both cellcellcontact and transwell cultures (Fig. 3A, 3C), although the sup-pression was more severe under cell-to-cell contact conditionsthan in the transwell system. There were no significantchanges in PGE2, IL-2, IL-4, IL-10, IL-12p70, TNFa, IFNc,or TGFb production observed in the supernatants of transwell

system (Supporting Information Fig. 3A3H). Furthermore,there was no difference of B cell proliferation index betweencocultured with autologous PDLSCs group and allogeneicPDLSCs group (Supporting Information Fig. 3I). The datasuggest that direct cell-to-cell contact contributed in large partto the mechanisms of hPDLSC-mediated suppression of B-cell proliferation.

hPDLSCs Suppress Immunoglobulins in B Cells

To investigate whether hPDLSCs and hBMSCs could inhibitB cell differentiation, B cells were cultured with or withouthPDLSCs or hBMSCs at a 1:1 ratio in the presence of CpGODN, rCD40L, anti-Ig, IL-2, IL-4, and IL-10. After 3 days,

nonadherent cells were collected and stained with anti-CD138mAb, which is an optimal marker for plasma cell detection. Asignificant decrease in CD138 expression was observed in Bcells cocultured with hPDLSCs or hBMSCs (*, p .003 and0.002, respectively; Fig. 4A). After 7 days, immunoglobulinsin the supernatants were determined by ELISA (Fig. 4B) andCBA assays (not shown). hPDLSCs or hBMSCs inhibited theproduction of IgM, IgG, and IgA at a B cell/mesenchymalcell ratio of 1:1. In contrast to their regulation of B-cell pro-liferation and differentiation, hPDLSCs and hBMSCs failed toaffect the expression of costimulatory molecules CD40, HLA-DR, CD86, and CD80 on B cells (Fig. 4C). These resultsindicate that hPDLSCs or hBMSCs suppress both B cell dif-ferentiation and immunoglobulin production.

Figure 1. Allogeneic miniature pig periodontal ligament stem cells (PDLSCs) regenerate periodontitis-related defects. (AD): Intraoral photo-graphs showed that, 12 weeks post-transplantation, only limited periodontal tissues were regenerated in the control group (A) and hydroxyapatite/tricalcium phosphate (HA/bTCP) group (B). Autologous(C) and allogeneic (D) pPDLSC-mediated periodontal tissue regeneration were nearly nor-mal. (EH): Computed tomography images revealed limited bone formation in the control group (E) and the HA/bTCP group (F) 12 weeks afterpPDLSC transplantation. Autologous pPDLSCs (G) and allogeneic pPDLSCs (H) mediated nearly complete alveolar bone regeneration. (IL):X-Ray imaging showed that, 12 weeks after pPDLSC transplantation, autologous (K) and allogeneic (L) pPDLSCs can regenerate more alveolarbone than the control group (I) or the HA/bTCP group (J). (MX): Hematoxylin/eosin staining in autologous (S) and allogeneic (V) pPDLSCgroups revealed new periodontal tissue regeneration (T, U, W, X) in the periodontal defect area. In the control (M) and HA/bTCP groups (P),deep periodontal pockets (N, O, Q, R)and few new bone and periodontal ligament fibers remained. (Yiiii): Clinical assessment of periodontal tis-sue regeneration. At week 4 and week 0, there was no significant difference among the four groups. However, 12 weeks post-transplantation, theprobing depth (PD), gingival recession (GR), and attachment loss (AL) were significantly improved in autologous and allogeneic pPDLSC-treat-ment groups compared with the control group and HA/bTCP group. There was also a significant difference between the control group and theHA/bTCP group. *, p


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Effects of hPDLSCs on B Cell Chemokine ReceptorExpression and Function

CXCR4, CXCR5, and CCR7, which function in the homingof B cells to secondary lymphoid organs, are important che-mokine receptors expressed constitutively by B cells [26, 27].The expression of CXCR4, CXCR5, and CCR7 in B cells sig-nificantly decreased after coculture with hPDLSCs orhBMSCs at a 1:1 ratio for 3 days in the presence of CpGODN, rCD40L, anti-Ig, IL-2, and IL-4 (CXCR4: *, p .036for hPDLSC coculture, *, p .023 for hBMSC coculture;

CXCR5: *, p .035 for hPDLSC coculture, *, p .021 for

hBMSC coculture; CCR7: *, p .002 for hPDLSC coculture,

*, p .0023 for hBMSC coculture) (Fig. 4D). Expression of

these chemokine receptors did not differ between B cells

cocultured with hPDLSCs and hBMSCs (Fig. 4D).To investigate whether these chemokine receptors could

affect the chemotaxis of activated B cells in response to theirligands (CXCL12, CXCL13, and CCL19, respectively), wedispensed purified B cells that had been cocultured with orwithout hPDLSCs or hBMSCs for 24 hours in the upper

Figure 2. Dynamic evaluation of humoral immunological parameters.(AD): There were no significant differences in the indicated time pointsregarding the percentage of CD20 or CD25 B cells in the allogeneic pig periodontal ligament stem cells (PDLSC) transplantation group atdifferent time points. (EG): Immunoglobulin (IgA, IgG, and IgM) in whole blood was nearly identical among the four test groups at week 4;at 1, 3, 5, 7 days, 2, 4, 8, and 12 weeks post-transplantation. (HJ): Immunoglobulin in gingival crevicular fluid was almost identical among thefour test groups at week 4; at 1, 3, 5, 7 days, 2, 4, 8, and 12 weeks post-transplantation. (KL) Immunoglobulin expression in regenerated peri-odontal tissues did not differ significantly between allogeneic and autologous PDLSC groups after 1 week(K) and 2 weeks (L) post-transplanta-

tion.(M): The expression of interleukin (IL)-6, programmed death (PD)-1 and PD-L1 was significantly higher in regenerated periodontal tissuesof the allogeneic PDLSCs group than in the autologous PDLSCs group at 1 week and 2 weeks after transplantation, whereas PD-L2 level did notchange. *, p < .05,

#*,p < .01. Abbreviations: HA, hydroxyapatite; TCP, tricalcium phosphate.



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transwell chamber, with CXCL12, CXCL13, CCL19 or me-dium alone added to the lower chamber. The results showedthat CXCL12-, CXCL13-, and CCL19-driven B cell chemo-taxis were significantly inhibited by culture with hPDLSCs orhBMSCs (Fig. 4E). These results suggest that hPDLSCs orhBMSCs could inhibit CXCR4-, CXCR5-, and CCR7-medi-

ated B-cell chemotaxis.

B Cells Promote IL-6 Production in hPDLSCs

We next investigated whether hPDLSCs affected cytokinerelease in B cells. The expression of PGE2, IL-2, IL-4, IL-6,IL-10, IL-12p70, TNFa, IFNc, and TGFb was determined incell supernatants in cultures containing hPDLSCs or hBMSCsalone, B cells alone, or B cells plus hPDLSCs or hBMSCs for3 days in the presence of CpG ODN, rCD40L, anti-Ig anti-bodies, IL-2, and IL-4. No significant differences in PGE2,IL-2, IL-4, IL-10, IL-12p70, TNFa, IFNc, or TGFb produc-tion were observed in the supernatants of any cultures (Sup-porting Information Fig. 3A3H). Interestingly, we observed asignificant increase in IL-6 in the supernatant of B cellscocultured with hPDLSCs or hBMSCs (Fig. 5A). In addition,

hPDLSCs and hBMSCs each secreted more IL-6 than B cellswhen they were cultured alone (Fig. 5A). Furthermore, invivo we also detected that the expression of IL-6 mRNA wassignificantly higher in regenerated periodontal tissues fromthe allogeneic PDLSC group than the autologous PDLSCgroup 1 and 2 weeks post-transplantation (Fig. 2M).

To define the cellular source of IL-6, ionomycin (250 ng/ml) and GolgiPlug protein transport inhibitor (1 ll/ml) wereadded to the culture. We analyzed intracellular IL-6 proteinwith flow cytometry in hPDLSCs or hBMSCs and B cellsafter 6 hours of coculture. We found that the frequency ofIL-6 cells in hPDLSCs or hBMSCs cocultured with B cellswas much higher than that in hPDLSCs or hBMSCs culturedalone (Fig. 5B). There were no significant differences betweenIL-6 B cells cocultured with hPDLSCs or hBMSCs and Bcells cultured alone (Fig. 5B). The data revealed that B cellspromote IL-6 production in hPDLSCs and hBMSCs.

hPDLSC-Derived IL-6 Protects B Cells fromApoptosis

Previous studies have shown that IL-6 is a pleiotropic cytokineproduced by lymphoid and nonlymphoid cells in response toseveral stimuli [28, 29]. The biological functions of IL-6include the regulation of proliferation, differentiation, and activ-ity of a wide variety of cell types [30] and participation in neu-roendocrine and immune system homeostasis. In this study, weshowed that the concentration of IL-6 in the supernatants ofhPDLSCs or hBMSCs cocultured with B cells was more thanin hPDLSCs, hBMSCs, or B cells cultured alone. Therefore,next we investigated the function of IL-6 in culture.

Neutralization of IL-6 with anti-IL-6 antibody had no sig-nificant effect on B-cell proliferation at 0, 50, 100, or 200 ng/ml (Fig. 5C). However, the percentage of apoptotic B cellsincreased substantially in a concentration-dependent mannerafter adding anti-IL-6 monoclonal antibodies (Fig. 5D). Theseresults indicate that hPDLSCs inhibit B-cell apoptosis viaIL-6.

Involvement of PD-1 in hPDLSC-MediatedImmunosuppression

PD-1, an inhibitory costimulatory molecule found on activatedT-cells, is involved in the regulation of immune responsesand peripheral tolerance [3133]. We studied whether PD-1 isinvolved in the hPDLSC-mediated immunoregulation of Bcell function. First, we detected that the expression of PD-1

and PD-L1 mRNA, but not PDL-2 mRNA, were significantlyhigher in regenerated periodontal tissues from the allogeneicPDLSC group than from the autologous PDLSC group 1 and2 weeks after transplantation in vivo in miniature pigs (Fig.2M). Next, we cultured B cells with hPDLSCs or hBMSCstogether with CpG ODN, rCD40L, anti-Ig antibodies, IL-2,

and IL-4. PD-1, PD-L1, and PD-L2 were significantlyincreased in hPDLSCs or hBMSCs cocultured with B cells ata 1:1 ratio (hPDLSCs: *, p .009, 0.0003, and 0.012, respec-tively; hBMSCs: *, p .0002, 0.017, and 0.048, respectively)(Fig. 5E).

To study the role of the PD-1 inhibitory pathway in medi-ating the suppression of cell proliferation by hPDLSCs orhBMSCs, we performed blocking experiments using monoclo-nal antibodies against PD-1, PD-L1, and PD-L2. The presenceof 0.5 lg/ml anti-PD-1 antibody restored approximately 90%of B-cell proliferation in coculture with hPDLSCs. Antibodieshad a dose-dependent effect on proliferation. The blockade ofPD-L2 did not produce any significant effect at 0.12, 0.25, or0.5 lg/ml. Adding anti-PD-L1 antibody (0.5 lg/ml) had only aminimal effect on the hPDLSC-mediated inhibition of B-cellproliferation. The simultaneous blockade of PD-L1 and PD-L2(0.5 lg/ml) produced no significant effect on the hPDLSC-mediated inhibition of cell proliferation. Similar results wereobtained when B cells were cocultured with hBMSCs (Fig.5F). Altogether, these results reveal a role for the PD-1 path-way in hPDLSC-mediated immunoregulation.

DISCUSSION

In this study, we provided novel evidence that allogeneicPDLSCs failed to activate humoral immunity in vivo in mini-ature pigs. We also showed that hPDLSCs inhibited humanB-cell proliferation, apoptosis, differentiation into antibodysecreting cells, and chemotaxis in vitro.

Periodontitis is characterized by progressive destruction ofperiodontal supporting tissue that results from microbial plaqueaccumulation, and it ultimately leads to tooth loss. In thisstudy, we observed that allogeneic PDLSCs, similar to autolo-gous PDLSCs, could effectively repair bone defects caused byperiodontitis in the miniature pig, which significantly improvedperiodontal tissue regeneration compared with the HA/TCPgroups. There were no significant alterations of immunoglobu-lins or B cell-associated activation markers (CD20, CD25) [34]between allogeneic and autologous PDLSC-treated miniaturepigs, suggesting lack of humoral immunological rejection inthe animals receiving allogeneic PDLSC transplantation. Thoseresults showed that allogeneic PDLSC transplantation will notcause humoral immunological response, which shed a light onfurther preclinical studies.

Next, we investigated underlying mechanisms of B celltolerance to allogeneic PDLSC transplantation. We showedthat hPDLSCs modulated B-cell functions, demonstrated bydecreases in proliferation, apoptosis, differentiation, chemo-taxis, and the expression of costimulatory molecules of Bcells in vitro. After culture with hPDLSCs, IgM-, IgG-, andIgA-producing B cells were significantly suppressed. BecauseB cell activation is influenced by T-cells that can be inhibitedby hPDLSCs [35] an indirect role through T-cell-mediatedsuppression of the PDLSC-mediated effect on B cells in vivocannot be completely excluded.

hBMSCs down-regulated HLA-DR, CD80 and CD86 cos-timulatory molecules in human mature myeloid dendritic cells[3638] but not in B cells [25]. In this study, we did not findhPDLSCs affect HLA-DR, CD40, CD80 and CD86


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Figure 3. (Continued)



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Figure 4. Human periodontal ligament stem cells (hPDLSCs) affect B-cell chemokine receptor expression and function, immunoglobulin pro-duction, and the expression of costimulatory molecules. (A):B cells cocultured with or without hPDLSCs at a 1:1 ratio in the presence of stimuliand stained with anti-CD138 antibody. Control B cells were cultured alone without stimuli. Results are shown as a percentage of positive cells.The differences are similarly related to changes in the expression of CD138 cells (plasmocytes) caused by coculturing with hPDLSCs or humanbone mesenchymal stem cells. n 5, *, p .003 and 0.002, respectively. (B): B cell expression of costimulatory molecules is unaffected byincubation with hPDLSCs. Purified B cells were cultured for 72 hours with or without hPDLSCs at ratios of 1:1 and 1:5, in the presence of CpGoligodeoxynucleotide (CpG ODN), recombinant CD40L (rCD40L), anti-Ig antibodies, interleukin (IL)-2, and IL-4. After culturing, B cells werestained with anti-HLA-DR, anti-CD40, anti-CD86, and anti-CD80 antibodies in combination with CD19 antibody and analyzed by flow cytome-try. Results are expressed as median percent positive cells, maximum value, and minimum value obtained from five different experiments. (C):hPDLSCs inhibit immunoglobulin production. B cells were incubated for 7 days without or with hPDLSCs at a 1:1 ratio in the presence of CpGODN, rCD40L, anti-Ig antibodies, IL-2, IL-4, and IL-10. Quantitation of IgM, IgG, and IgA in culture supernatants was evaluated by ELISA. n 5. *, p 0 .036 for IgA, p .0001 for IgG, and p .013 for IgM. (D): Purified B cells were cultured 24 hours without or with hPDLSCs at1:1 with CpG ODN, rCD40L, anti-Ig antibodies, IL-2, and IL-4. After culture, B cells were stained with anti-CXCR4, anti-CXCR5, or anti-CCR7 antibodies in combination with CD19 antibody, and analyzed by flow cytometry. Results are expressed as median percent positive cells

obtained from five different experiments. *, p .036 for CXCR4 and 0.035 for CXCR5 at a B-cell/hPDLSC 1:1 ratio; p .002 for CCR7 at a1:1 ratio. (E): Chemotaxis of activated B cells cultured with or without hPDLSCs at a 1:1 ratio in response to CXCL12, CXCL13, CCL19 (theligands of CXCR4, CXCR5, and CCR7, respectively), or medium alone. Migrated B cells were identified by CD19 staining. n 5. *, p .021for CXCL12 at the B-cell/hPDLSC 1:1 ratios, and *, p .032 for CXCL13 at a 1:1 ratio. Abbreviations: hBMSCs, human bone mesenchymalstem cells; HLA-DR, human leukocyte antigen - DR; hPDLSCs, human periodontal ligament stem cells; Sti, Stimulation.

Figure 3. Human periodontal ligament stem cells (hPDLSCs) inhibit B-cell proliferation and apoptosis.(A): Flow cytometric analysis confirmedthat purified carboxyfluorescein succinimidyl ester (CFSE)-labeled B cells (from peripheral blood from healthy donors) were cocultured for 3 dayswith allogeneic hPDLSC suspensions at 5:1, 1:1, 1:1 (Transwell), and 1:5 ratios with or without CpG oligodeoxynucleotide (CpG ODN), recombi-nant CD40L (rCD40L), anti-Ig antibodies, interleukin (IL)-2, and IL-4. Cell proliferation was assessed by flow cytometric analysis. (B): Statisticalanalysis revealed that hPDLSCs inhibited dose-dependent B cell proliferation. *, p < .05,

#*, p < .01, n 5. (C): hPDLSCs and B cells wereseeded 1:1 in 12-well plates on the opposite sides of a 4-lm pore-size polycarbonate membrane (Transwell) and cultured for 3 days. Aliquots ofthe same hPDLSC and B-cell suspensions were cocultured 1:1 for 3 days. Control B cells were cultured alone. Stimuli were CpG ODN, rCD40L,anti-Ig, IL-2, and IL-4. B-cell proliferation was assessed by CFSE. Results showed that hPDLSCs, similar to human bone mesenchymal stem cells,inhibited B cell proliferation under both cell-cell contact and transwell cultures, although the suppression was more severe under cell-to-cell contactconditions than in the transwell system. *, p < .05,

#*,p < .01, n 5. (D): B-cells cultured for 24 hours and 72 hours with or without hPDLSCs

(1:1) in the presence of stimuli were double stained with propidium iodide and Annexin V and analyzed by flow cytometry. (E):Statistical analysisshowed that hPDLSCs inhibited B cell apoptosis. *, p < .05,n 5. Abbreviations: AV FITC, FITC conjugated Annexin V; CFSE, carboxyfluores-cein succinimidyl ester; hBMSCs, human bone mesenchymal stem cells; hPDLSCs, human periodontal ligament stem cell; Sti, Stimulation.



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Figure 5. The mechanism of human periodontal ligament stem cells (hPDLSCs) effect on B-cells proliferation and apoptosis. (A): The inter-leukin (IL)-6 concentration significantly increased with or without hPDLSCs; with or without CpG oligodeoxynucleotide (CpG ODN), recombi-nant CD40L (rCD40L), anti-Ig antibodies, IL-2, and IL-4; and without B cells in the Transwell culture system. However, it was less than theIL-6 concentration in cell-to-cell contact culture. n 5, *, p < .05. (B): B cells were cocultured with or without hPDLSCs and with or withoutCpG ODN, rCD40L, anti-Ig antibodies, IL-2, and IL-4 in the presence of Gorky protein inhibitors. B cells were fixed, permeabilized, and stainedwith CD19 and anti-IL-6mAb. Results show that IL-6 was secreted by hPDLSCs (n 5, *, p < .05). (C): The neutralizing monoclonal antibodyagainst IL-6 failed to restore B-cell proliferation. (D): The percentage of apoptotic B cells increased in a concentration-dependent manner after

adding anti-IL-6 monoclonal antibodies to the cultures at 0, 50, 100, or 200 ng/ml. (E): hPDLSCs were cocultured for 3 days without or with Bcells at 1:1 without or with CpG ODN, rCD40L, anti-Ig antibodies, IL-2, and IL-4. Post-culture, hPDLSCs were stained with monoclonal antibod-ies against programmed death (PD)-1, PD-L1, and PD-L2. Expression of PD-1, PD-L1, and PD-L2 by human bone mesenchymal stem cells(hBMSCs) under different culture conditions was analyzed by flow cytometry. PD-1, PD-L1, and PD-L2 expression were significantly increasedin hPDLSCs and hBMSCs in the coculture system (hPDLSCs: *, p .009, 0.0003, and 0.012, respectively; hBMSCs: *, p .0002, 0.017, and0.048, respectively). (F): We performed blocking experiments using monoclonal antibodies against PD-1, PD-L1, and PD-L2. Anti-PD-1 antibody(0.50lg/ml) restored approximately 90% of B-cell proliferation in coculture with hPDLSCs, and cell proliferation was rescued by anti-PD-1 in adose-dependent fashion. The blockade of PD-L2 did not produce any significant effect at the concentration of 0.12, 0.25, and 0.5 lg/ml. Therewas only a little significant effect by adding anti-PD-L1 antibody at the concentration of 0.5 lg/ml on the hPDLSC-mediated inhibition of cellproliferation. Simultaneous blockade of PD-L1 and PD-L2 did not produce any significant effect at 0.12 or 0.25 lg/ml. There was a small butsignificant effect on the hPDLSC-mediated inhibition of cell proliferation at 0.5 lg/ml. In coculture with hBMSCs, B-cell proliferation wasrestored to approximately 60% of B-cell proliferation. B-cell proliferation was rescued by anti-PD-1, although not in a dose-dependent fashion;maximum rescue of B-cell proliferation was observed at 0.5 lg/ml. Although the simultaneous blockade of PD-L1 and PD-L2 partly rescuedB-cell proliferation, it did not significantly affect the BMSC-mediated inhibition of cell proliferation. Abbreviations: hBMSCs, human bone mes-enchymal stem cells; hPDLSCs, human Periodontal ligament stem cells; IL-6, interleukin-6; PD, programmed death; Sti, Stimulation.



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expressions on B cells, suggesting that the antigen-presentingfunction of B cells was unsuppressed. However, we showedthat CXCR4, CXCR5, and CCR7 in B cells were down-regu-lated after incubation with hPDLSCs, paralleled by decreasedB-cell chemotaxis in response to CXCL12, CXCL13, andCCL19, respectively.

In previous studies, hBMSC-mediated immune suppressionof activated B cells has been attributed to the secretion of anti-proliferative soluble factors, such as PGE2, CCL2, CCL7, Blimp-1, and PD-1 and its ligands, as well as cell-to-cell contact [3842], albeit not unanimously [34], and CD73, which is expressedin BMSCs, produces adenosine which can directly inhibit B cells[43, 44]. In this study, we demonstrated that hPDLSCs sup-pressed B-cell proliferation through PD-1 and PD-L1 interactionin vitro. This mechanism differs from that of hBMSCs, whichsuppressed B-cell proliferation through the interaction of PD-1with PD-L1 and PD-L2. Although our findings indicated thathPDLSC-mediated inhibition of B-cell proliferation occurredthrough cell-to-cell contact, the detailed mechanisms involvedrequire additional investigation. Similarly to observations regard-ing T-cells [18], B cells failed to proliferate but did not undergoapoptosis with hPDLSC treatment. On the contrary, our datashowed that hPDLSCs inhibited B cell apoptosis by secreting IL-6. We also observed that the expression of IL-6, PD-1, and PD-L1 mRNAs (but not PD-L2 mRNA) was significantly higher inregenerated periodontal tissues from the allogeneic PDLSC groupthan the autologous PDLSC group 12 weeks post-transplanta-tion. After transplantation, Th1, Th17 and the high level ofIL-17, TNF-a and IFN-c in the periodontitis tissue canincrease the expression of PD-L1 on MSCs, which inhibitsthe differentiation of B cells via PD-1 /PD-L1 pathway [45].All MSCs were uniformly positive for HLA-ABC and lackedthe expression of HLA-DR and the costimulatory molecules(e.g., CD40, CD80, CD86, CD134, and CD252) required forfull T-cell activation [46], and in our previous study weshowed that hPDLSCs displayed low immunogenicity [18].However, after transplantation, a small amount of allogeneic

PDLSCs might be presented to B cells as antigen by anti-gen-presenting cells, and these B cells might be stimulatedand express IL-6 and B7, which activated the PD-1/PD-L1expression of MSCs in periodontal tissue. Previous studiessuggest that activated human B cells can circulate throughoutthe body [47], so the B cells in inflamed periodontal tissuecan be existed in situ or migrated from lymph nodes andother inflammatory tissues. After treatment, the allogeneicMSCs migrated followed CXCL12, CXCL13, and CCL19density [11, 48] toward the site of inflamed area, suppressedthe chemotaxis in B cells. Meanwhile, the proliferation of en-dogenous B cells were also inhibited through cell-to-cellcontact.

The therapeutic potential of BMSCs is currently beingexplored in a number of phase I/II and III clinical trials [48],

and a BMSCs product, Prochymal (Osiris Therapeutics Inc.),has now been used for clinical treatment. It has been reportedthat PDLSCs possessed greater proliferative potential thanBMSCs. PDLSCs have also same potential of single-colony-strain generation, multipotential differentiation [49], andexhibit the same immune property to what cells? [18]. More-

over, PDLSCs can regenerate cementum/periodontal-ligament-like tissue [16], thus PDLSCs were more suitable for peri-odontal tissue regeneration.

SUMMARY

In summary, allogeneic PDLSCs can regenerate periodontaltissues in vivo without evidence of humoral immuneresponses. hPDLSCs interfere with human B-cell functionby acting at multiple levels, including but not limited toproliferation, differentiation into antibody-producing cells,and chemotaxis. Thus, the inhibition of B-cell functions inconcert with the suppression of T-cell activation furthersupports that the use of allogeneic hPDLSCs might be a

promising approach for tooth regeneration and periodontalligament reconstruction in patients with periodontaldiseases.

ACKNOWLEDGMENTS

This work was supported by the grant from Beijing Munic-ipal Committee for Science and Technology(Z121100005212004; to S.W.); the National Basic ResearchProgram of China (2010CB944801; to S.W.), the FundingProject for Academic Human Resources Development inInstitutions of Higher Learning Under the Jurisdiction of

Beijing Municipality (PHR20090510; to S.W.), NationalNatural Science Foundation of China (NSFC NO.81070799;to G.D.), and the Funding Project to Science Facility inInstitutions of Higher Learning Under the jurisdiction ofBeijing Municipality (PXM 2009-014226-074691; to S.W.).W.C. was supported by the Intramural Research program ofthe National Institute of Dental and Craniofacial Research,National Institutes of Health.

DISCLOSURE OFPOTENTIALCONFLICTS OFINTEREST

The authors indicate no potential conflicts of interest.

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