of July 28, 2018.This information is current as

Dendritic Cells Regulate T Cell Responses−CD19+B220+Liver-Derived DEC205

Fung and Shiguang QianNalesnik, Mark S. Schlissel, Anthony J. Demestris, John J.

A.Chen, Wei Li, Liangfu Wang, Simon C. Watkins, Michael Lina Lu, C. Andrew Bonham, Xiaoyan Liang, Zongyou

http://www.jimmunol.org/content/166/12/7042doi: 10.4049/jimmunol.166.12.7042

2001; 166:7042-7052; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/12/7042.full#ref-list-1

, 39 of which you can access for free at: cites 66 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

by guest on July 28, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Liver-Derived DEC2051B2201CD192 Dendritic Cells RegulateT Cell Responses1

Lina Lu, 2* C. Andrew Bonham,* Xiaoyan Liang,* Zongyou Chen,* Wei Li,* Liangfu Wang,*Simon C. Watkins,† Michael A. Nalesnik,‡ Mark S. Schlissel,§ Anthony J. Demestris,‡

John J. Fung,* and Shiguang Qian*

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse livernonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directingdifferentiation of naive allogeneic T cells. After culture, such cells are DEC-205bright B2201CD11c2CD192, and negative for T(CD3, CD4, CD8a), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC2051B2201

CD192 cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrange-ments, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR dueto extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide,benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC2051B2201D192 cellsrelease both IL-10 and IFN-g, small amounts of TGF-b, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1cells. Expression of IL-10 and IFN-g, but not bioactive IL-12 in liver DEC2051B2201CD192 cells was demonstrated by RNaseprotection assay. In vivo administration of liver DEC2051B2201CD192 cells significantly prolonged the survival of vascularizedcardiac allografts in an alloantigen-specific manner. The Journal of Immunology,2001, 166: 7042–7052.

T cell differentiation is crucial to the outcome of an im-mune response. Early in the process of activation, T cellsare committed to develop into one of several functionally

distinct subsets, including Th1, Th2, and the recently described Tregulatory (Tr)3 cells. Tr cells may play a critical role in the gen-eration and maintenance of tolerance.

Several varieties of Tr cells have been described, each withunique, albeit somewhat nebulous characteristics. Thus, a numberof definitions of Tr cells exist in the literature (1–3). Type 1 Tr(Tr1) cells are a subset characterized by their unique profile ofcytokine production. Tr1 cells produce high levels of IL-10, mod-erate amounts of TGF-b and IFN-g, but no IL-4 or IL-2. Theyexert immunoregulatory or suppressive effects (1–4).

T cell differentiation is regulated by the local microenvironment.Hence, the property of Ags encountered by the T cell, and theexpression of costimulatory molecules and cytokines by APCsdrive T cell differentiation. In vitro, IL-12 drives Th1 (5, 6),whereas IL-4 promotes Th2 differentiation (7, 8). Similarly, thegeneration of Tr1 cells is driven by IL-10 (1). The stimulus con-

trolling T cell differentiation during an in vivo immune response isless clear.

Dendritic cells (DC) are uniquely suited for activation of naiveT cells (9). Recent data suggest that different DC subsets provideT cells with selective signals that guide either Th1 or Th2 differ-entiation. In mice, DC have been classified into myeloid and lym-phoid subsets according to their phenotype and their developmentfrom distinct precursors (10–14). These subsets of DC share anumber of distinct properties, including dendritic morphology, theability to migrate, and expression of a range of molecules requiredfor activation of naive T cells. However, they differ in their reg-ulation of the immune response. Thus, myeloid DC usually initiateimmune responses, and typically induce Th1 differentiation. Incontrast, the so-called lymphoid DC propagated in response toIL-3, while capable of activating lymphocytes, may also limit Tcell proliferation by inducing Fas-mediated apoptosis and inhibit-ing cytokine production (15–18). Analogous to mice, humans mayalso contain two DC types developed from distinct precursors.DC1, propagated in response to GM-CSF from peripheral bloodmonocytes, produce high levels of IL-12 and induce Th1 differ-entiation. On the other hand, DC2 propagated from blood or tonsilplasmacytoid T cells in response to IL-3, drive Th2 differentiation(19, 20). Furthermore, repetitive stimulation with allogeneic im-mature DC induces IL-10-producing, nonproliferating T cells withregulatory properties (3).

In studies designed to assess liver-derived DC and their functionin vitro and in vivo, we have identified a novel cell populationpropagated from normal mouse liver nonparenchymal cells (NPC)in response to IL-3 and anti-CD40 mAb. These cells exhibit DCmorphology and express the DC marker DEC205 (21). They alsobear the B220 Ag, a marker of cell activation typically associatedwith B cells, but do not express the B cell Ag CD19. They activateT cells, with subsequent induction of T cell apoptosis. A smallerproportion of T cells is stimulated to release IFN-g, IL-10, and

*Thomas E. Starzl Transplantation Institute and Department of Surgery,†Departmentof Cell Biology and Physiology, and‡Department of Pathology, University of Pitts-burgh Medical Center, Pittsburgh, PA 15213; and§Department of Molecular and CellBiology, University of California, Berkeley, CA 94720

Received for publication December 22, 2000. Accepted for publication April 3, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by National Institutes of Health Grant DK 29961 andJuvenile Diabetes Foundation International Grant P1893135.2 Address correspondence and reprint requests to Dr. Lina Lu, Thomas E. StarzlTransplantation Institute and Department of Surgery, University of Pittsburgh Med-ical Center, E1554 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA15213. E-mail address: lul@msx.upmc.edu3 Abbreviations used in this paper: Tr, T regulatory; BM, bone marrow; DC, dendriticcell; MST, median survival time; NPC, nonparenchymal cell; S:R, stimulator:re-sponder; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

TGF-b, cytokines resembling a Tr1 cell phenotype. The propa-gated cells exhibit Ig gene rearrangements consistent with devel-oping B cells, but lack surface expression of Ig. After injection intoallogeneic recipients, they migrate to spleen and dramatically pro-long a subsequent cardiac allograft.

Materials and MethodsAnimals

Male C57BL/10 (B10; H-2b), C3H (H-2k), and BALB/c (H-2d) mice werepurchased from The Jackson Laboratory (Bar Harbor, ME) and used at8–12 wk of age. Animals were maintained in the specific pathogen-freefacility of the University of Pittsburgh Medical Center (Pittsburgh, PA) andprovided with Purina rodent chow (Ralston Purina, St. Louis, MO) and tapwater ad libitum.

Isolation of NPC from liver

Livers were perfused in situ with collagenase solution, followed by furtherex vivo digestion. The NPC fraction was then isolated by centrifugationover a Percoll gradient (Sigma, St. Louis, MO), as described previously(22).

Propagation of DEC2051, B2201, CD192 cells from livers

Liver NPC were depleted of T, B, NK, granular cells, and macrophages bycomplement-dependent lysis using a mAb mixture comprising anti-CD3,CD19, NK1.1, CD14, Gr-1 (all Abs from PharMingen, San Diego, CA),and low toxicity rabbit complement (Accurate Chemical and Scientific,Westbury, NY). Thereafter, 23 106 lineage-negative cells were cultured in2 ml of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplementedwith antibiotics and 10% (v/v) FCS (referred to subsequently as completemedium), and mouse rIL-3 (10 ng/ml; BioSource International, Camarillo,CA), plus anti-CD40 mAb (2 ng/ml; PharMingen) in flat-bottom 24-wellculture plates for 5–7 days. Nonadherent cells released from clusters wereharvested for further characterization. For comparative purposes, maturemyeloid DC propagated from bone marrow (BM) in GM-CSF plus IL-4(referred to subsequently as BM IL-4 DC) and immature myeloid DC prop-agated from liver NPC in GM-CSF alone (referred to subsequently as liverGM-CSF DC), as described elsewhere (22, 23), were used. Briefly, BMcells or liver NPC were cultured in 24-well plates (23 106/well) in com-plete medium containing both mouse rGM-CSF (4 ng/ml) and rIL-4 (1000U/ml) (both from Schering-Plough, Kenilworth, NJ) or GM-CSF alone for5–7 days. The selection and purification procedures were similar to thosereported initially by Inaba et al. (24) and modified by Lu et al. (22, 23).

Flow cytometry

Cell surface Ag expression was analyzed by cytofluorography using anEpics Elite flow cytometer (Coulter, Hialeah, FL). FITC- or PE-conjugatedmAbs were obtained from PharMingen, except for anti-DEC-205 mAb(generously provided by R. M. Steinman, The Rockefeller University, NewYork, NY). For intracellular cytokine detection, cells were incubated inbrefeldin A (10mg/ml; Sigma) for 5 h, then washed with 1% saponin/1%FCS/PBS, as described previously (25). Double staining was performedusing FITC- or PE-conjugated anti-H-2Kb, anti-IL-10, or anti-IFN-gmAbs. Cells were then washed with 1% FCS/PBS and resuspended in 1%formaldehyde before analysis. Appropriate isotype- and species-matchedirrelevant mAbs were used as controls.

Detection of apoptosis

T cells were stained with PE-conjugated anti-CD3e, anti-CD4, or anti-CD8a mAb, and DNA strand breaks were identified by TUNEL. Followingsurface CD3, CD4, or CD8 staining, cells were fixed in 4% paraformal-dehyde and permeabilized with 0.1% Triton X-100 and 0.1% sodium ci-trate. TUNEL reaction mixture of the Cell Death Detection kit (RocheDiagnostics, Indianapolis, IN) was then added according to the manufac-turer’s instructions. Cells incubated with label solution in the absence ofterminal transferase were used as negative controls. Quantitative analysiswas performed by flow cytometry, with 5000 events acquired from eachsample. For identification of apoptotic cells in cytospin preparations, Tcells activated by DC were processed for immunocytochemical detectionof incorporated biotin-dUTP by peroxidase-labeled avidin, followed by anenzyme reaction using aminoethylcarbazole as the substrate, as describedelsewhere (26).

Mixed leukocyte reaction

To determine the allostimulatory capacity of DC, one-way MLR were per-formed withg-irradiated (20 Gy) DC or spleen cells from B10 (allogeneic)or C3H (syngeneic) mice as stimulators and nylon wool-purified C3Hspleen T cells (23 105) as responders. Cultures (200ml) were establishedin triplicate in 96-well round-bottom microculture plates and maintained incomplete medium in 5% CO2 in air at 37°C for 3–4 days. [3H]TdR (1mCi/well) was added for the final 18 h of culture, and incorporation of[3H]TdR into DNA was assessed by liquid scintillation counting in anautomated counter. Results are expressed as mean cpm6 1 SD. In apo-ptosis inhibition experiments, a common caspase inhibitor peptide, benzy-loxycarbonyl-Val Ala-Asp-fluoromethyl ketone (zVAD-fmk; Alexis,San Diego, CA), was added (100mM) at the beginning of the MLR culture.DMSO served as a control.

Cytokine and NO quantitation

IL-2, IFN-g, IL-4, IL-10, IL-12, TNF-a, and TGF-b levels in supernatantsof MLR or DC cultures were quantitated using ELISA kits (BioSourceInternational), with sensitivity limits of 20–25 pg/ml, as described (25). Astandard curve using recombinant cytokine was generated for each assay.NO levels were determined by the colorimetric Griess reaction that detectsthe stable end product nitrite, as described (26).

RNase protection assay

Total RNA was extracted from DC by the guandinium isothiocyanate-phenol-chloroform method using TRI reagent (Sigma), as described (27).Cytokine mRNA expression was determined using the RiboQuant multi-probe RNase protection assay system (PharMingen) following the manu-facturer’s instruction. Briefly, 5mg of total RNA was hybridized to32P-labeled RNA probes overnight at 56°C, followed by treatment with RNasefor 45 min at 30°C. The murine L32 and GAPDH riboprobes were used ascontrols. Protected fragments were submitted to electrophoresis through a7 M urea/5% polyacrylamide gel and then exposed to Kodak X-OMATfilm (Kodak, Rochester, NY) for 72 h.

DNA PCR assay for Ig rearrangement

DNA was prepared for PCR by lysing cells in 200ml of PCR lysis buffer(10 mM Tris, pH 8.4, 50 mM KCl, 2 mM MgCl2, 0.45% Nonidet P-40,0.45% Tween 20, and 60mg/ml proteinase K), incubating them at 55°C for1 h, and then inactivating the protease by heating to 95°C for 10 min. DNAat a concentration of 5000 genomes/ml was used for PCR. PCR (50ml)were performed as described previously (28). Thirty cycles of amplificationwere performed, after which one-fifth of each reaction was analyzed on a1.4% agarose gel in Tris-borate buffer. The gel was blot transferred to anylon membrane and probed with32P-labeled DNA from the appropriate IgC region. Primer sequences are as published elsewhere (28). Germlinealleles were detected using a primer (Mu0) 322 nt 59to JH1. DH R and Lprimers are oligonucleotide mixtures degenerate at two and three positions,respectively, and homologous to all members of theDfl16andDsp2D genefamilies. D to J rearrangements were detected as amplified fragments of;1033, ;716, or ;333 nt depending on whether JH1, JH2, or JH3 wasrearranged. To detect V to DJ rearrangements, a mixture of three differentdegenerate oligonucleotides homologous to conserved framework region 3sequences of three VH gene families (VH7183, VH558,andVHQ52) and theJ3 primer was used. This results in amplified VDJ rearrangements of;1058,;741, or;358 nt. PCR products were detected by hybridizationwith appropriate Ig gene probes. Both DJ and VDJ rearrangements resultin loss of Mu0 sequence and its amplification product. V to DJ rearrange-ment events result in loss of all of the DH L primer target sequences andamplified DJ fragments.

In vivo migration

Cells propagated from B10 mice were injected s.c. (53 105 cells in 50ml)into a hind footpad of normal allogeneic C3H recipients. Animals weresacrificed in groups of three at days 1, 2, 3, and 7 after injection. Thedraining popliteal lymph node, thymus, and spleen were removed, embed-ded in Tissue-Tek OCT compound (Miles, Elkart, IN), and frozen at280°C. Cryostat sections (4mm) were air dried at room temperature over-night for further processing.

Immunohistochemistry

B10 MHC class II1 cells were identified in cryostat sections or cytospinpreparations using biotinylated mouse IgG2a anti-mouse I-Ab (PharMin-gen) in an avidin-biotin-alkaline phosphatase complex (ABC) staining pro-cedure. Isotype- and species-matched irrelevant mAb were used as control.

7043The Journal of Immunology

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

Donor MHC class II1 (I-Ab1) cells were counted in 100 high-power fields,and the data were expressed as number of I-Ab1 cells per high-power field.

Heterotopic vascularized heart transplantation

Surgical procedures were performed under methoxyflurane (Medical De-velopment, Springvale, Australia) inhalation anesthesia. Cardiac anasto-moses to the abdominal aorta and inferior vena cava were performed asdescribed previously (29). The function of the donor heart was monitoreddaily by abdominal palpation. Rejection was defined as total cessation ofcontraction, which was confirmed by histological examination.

Statistical analyses

Graft survival times between groups of transplanted animals were com-pared using the Mann-WhitneyU test. A p ,0.05 was considered to bestatistically significant.

ResultsGeneration of DEC2051B2201CD192 cells from liver NPC

NPC were isolated from livers of B10 mice. Approximately 7–83106 cells were obtained from each liver, with,5% hepatocytecontamination. An Ab mixture and complement were used to de-plete CD41, CD8a1, CD141, CD191, NK1.11, and Gr-11 cells.Cells were then cultured in complete medium containing IL-3 for3–4 days, and clusters of proliferating cells were noted (Fig. 1A).Addition of anti-CD40 mAb induced the formation of long den-dritic process on these cells. Following 3–4 additional days inculture, the cells detached from the adherent clusters. By 6–8days,;3 3 106 such cells were obtained from each mouse liver.

Morphologically, the cells displayed characteristics of DC, in-cluding irregular-shaped eccentric nuclei, a paucity of prominentcytoplasmic granules, and extended dendrites (Fig. 1B). Transmis-sion electron microscopy further delineated the delicate cytoplas-mic processes, an abundance of mitochondria, and the absence ofelectron-dense granules (Fig. 2A). The typical dendritic veils orpseudopodia were observed under scanning electron microscopy(Fig. 2, B andC).

Immunophenotypic analysis demonstrated high expression ofCD45, MHC class I, MHC class II, costimulatory molecules(CD40, CD80, and CD86), and the lymphoid DC marker DEC-205(Fig. 3A). The myeloid DC marker CD11c was absent. Addition-ally, the cells did not express Ags associated with myeloid cells(CD13, CD11b, or CD14), T cells (CD3e, CD4, and CD8a), or NKcells (NK1.1). Of interest, the cells expressed B220, an Ag typi-cally present on B cells, but lacked the B cell-restricted moleculeCD19 (Fig. 3B).

By contrast, liver GM-CSF DC expressed the myeloid lineagemolecules CD11b, CD13, and CD14, as well as the myeloid DCmarker CD11c (Fig. 3A), as described (22). They exhibited onlylow levels of DEC-205. MHC and costimulatory molecule expres-sion were low (Fig. 3A), which was consistent with an immature

phenotype. Further stimulation with anti-CD40 mAb, Flt-3 ligand,or extracellular matrix protein induced partial or full maturation(22, 30). Thus, two distinct subsets of cells bearing molecules as-sociated with Ag presention can be propagated from precursorspresent in liver NPC in response to different cytokines (IL-3/CD40ligand vs GM-CSF). BM IL-4 DC show a mature myeloid DCphenotype (data not shown), as reported (23).

Ig gene rearrangement and expression in liver-derivedDEC2051B2201CD192 cells

The expression of B220 by cells derived from liver NPC in re-sponse to IL-3 and CD40 ligation raises the possibility that theyderive from B cells. Rearrangement of Ig genes occurs relativelyearly in B cell development, and are detectable in all but the ear-liest precursors. DNA was isolated from purified liver-derivedDEC2051B2201CD192cells (sorted by flow cytometry to achieve

FIGURE 1. Cells propagatedfrom normal B10 (H-2b) liver NPCwith IL-3 and anti-CD40 mAb.A,Cell clusters at 48 h of culture.B,Immunocytochemical staining forMHC class II (I-Ab) expression ofcells cultured for 6 days, displayingtypical DC morphology with long,thin, and beaded processes.

FIGURE 2. Ultrastructure of liver NPC-derived cells propagated with IL-3and anti-CD40 mAb.A, Transmission electron micrographs of cells culturedfor 6 days demonstrate numerous, extensive cytoplasmic processes, irregularlyshaped nuclei, numerous mitochondria, and a paucity of paracrystalline cyto-plasmic granules.B andC, Scanning electron micrographs of cells showingveils (3-day (B) and 5-day (C) culture). Bars, 1mm.

7044 DEC2051, B2201, CD192 CELLS REGULATE T CELL DIFFERENTIATION

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

FIGURE 3. A, Flow cytometric analysis of cell sur-face Ag expression on liver-derived DEC2051B2201

CD192 cells propagated with IL-3 and anti-CD40 mAb(filled histograms) compared with liver GM-CSF DC(open histograms). Appropriate Ig isotype controls areshown as dotted profiles.B, Liver DEC2051B2201

CD192 cells were double stained with PE anti-DEC205and FITC anti-B220 or FITC anti-CD19. Data are rep-resentative of three separate experiments.

FIGURE 4. A, DNA PCR analysis for Ig gene rear-rangements in liver-derived DEC2051B2201CD192

cells. DNA was isolated from CD11c1 BM-IL-4 DC(lane 1), or liver DEC2051B2201CD192 cells (lanes 2and3, representing samples from two experiments) pu-rified by flow cytometry.Lane 4, DNA ladder. Thymusand spleen cells are represented inlanes 5and 6. ARAG-deficient pro-B cell line 63-12 is shown inlane 7.Lane 8, Cell-free negative control. D to JH, V to DJH,and V to Jk gene rearrangements were identified inDEC2051B2201CD192 cells.B, Flow cytometric anal-ysis of Ig expression on liver DEC2051B2201CD192

cells. Cells were double stained with anti-B220 PEor DEC205 PE and anti-CD19, anti-IgG, anti-IgM,anti-Igk, or anti-Igl FITC mAb, showing theseDEC2051B2201CD192 cells do not express IgG andIgM, but express low Igk. The data are representative ofthree separate experiments.

7045The Journal of Immunology

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

purity .99%), and analyzed by PCR for Ig gene rearrangements.D to JH, V to DJH, and V to Jk gene rearrangements were identifiedin the cells (Fig. 4A). A similar pattern was noted in splenocytesrich in mature B cells. In contrast, myeloid DC had only Ig heavychain DJ rearrangements. This is not surprising, as many T cellsand other myeloid lineage cells have similar rearrangements. How-ever, the myeloid DC lack VDJ and VJk alleles (Fig. 4A).

To determine expression of Ig proteins, the DEC2051B2201

CD192 cells were stained with mAbs specific to mouse IgG, IgM,Igk, or Igl. The cells lacked expression of IgG, IgM, and Igl. Asmall proportion of cells expressed low levels of Igk l (Fig. 4B).Expression ofk light chain is normally found in immature B cellsin conjunction with am-heavy chain to form IgM (31). These datasuggest that DEC2051B2201CD192 cells have developed from B

FIGURE 5. A, [3H]TdR uptake by T cells inMLR. C3H (H-2k) splenic T cells were culturedwith g-irradiated B10 (H-2b) spleen cells, maturemyeloid DC (BM IL-4 DC), immature myeloid DC(liver GM-CSF DC), or liver-derived DEC2051

B2201CD192 cells for 3 days at various S:R ratios.B, Cytocentrifuge preparations of cells from 3-dayculture were stained by in situ nick-end labeling(TUNEL). T cells cultured with liverDEC2051B2201CD192 cells at T:DC ratio of 10:1demonstrated higher levels of apoptosis than T cellscultured with BM IL-4 DC or liver GM-CSF DC.Original magnification,3100. C, Allostimulatoryactivity of liver-derived DEC2051B2201CD192

cells was restored by addition of the commoncaspase inhibitor zVAD-fmk (100mM) at the be-ginning of 3-day MLR. Results are expressed asmean cpm6 SD of triplicate cultures and are rep-resentative of three experiments.

7046 DEC2051, B2201, CD192 CELLS REGULATE T CELL DIFFERENTIATION

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

cell precursors (pro-B or pre-B), but have arrested or diverged atsome point before becoming immature B cells.

Allostimulatory capacity of liver-derived DEC2051B2201

CD192 cells

The allostimulatory capacity of DEC2051, B2201, CD192 cellsderived from B10 liver NPC was determined in a one-way MLR.Mature myeloid DC (BM IL-4 DC) stimulated vigorous allogenic Tcell proliferation, whereas liver-derived DEC2051B2201CD192

cells induced very little T cell proliferation, as determined by thy-midine uptake. The allostimulatory capacity was similar to thatseen with immature myeloid DC propagated from liver in responseto GM-CSF (liver GM-CSF DC) (Fig. 5A) (22). Low T cell pro-liferation after stimulation by liver GM-CSF DC was expected dueto low expression of MHC and costimulatory molecules (Fig. 3A)(22). However, the DEC2051B2201CD192 cells expressed highlevels of these molecules (Fig. 3A) and would be expected tostimulate a brisk T cell response. Direct inspection of the T cellsover the course of the MLR revealed evidence of T cell death. After2 days in culture, similar levels of T blasts developed in re-sponse to BM IL-4 DC and liver-derived DEC2051B2201 CD192

cells. Few blasts were observed in T cells responding to liverGM-CSF DC. However, T cells stimulated by liver-derivedDEC2051B2201CD192 cells rapidly died, as determined by insitu TUNEL staining. After 3 days in culture, a large number ofapoptotic cells mixed with large blast cells were visible in T cellsstimulated by liver DEC2051B2201CD192 cells (Fig. 5B). Incontrast, few T cells stimulated by BM IL-4 DC or liver GM-CSFDC exhibited evidence of cell death. The low thymidine uptakeby T cells stimulated by liver-derived DEC2051B2201CD192

cells, despite the appearance of T cell blasts, must therefore be dueto rapid apoptosis of activated T cells. Indeed, inhibition ofapoptosis by addition of the caspase inhibitor peptide zVAD-fmk(32) restored T cell proliferation induced by liver-derivedDEC2051 B2201CD192 cells (Fig. 5C).

Liver-derived DEC2051B2201CD192 cells induce T cellapoptosis

To confirm that T cells stimulated by allogenic liver DEC2051

B2201CD192 cells undergo apoptosis, C3H splenic T cells cul-tured with various DC subtypes from B10 donors for 1–3 dayswere stained by TUNEL and anti-CD3, anti-CD4, or anti-CD8mAbs. Two-color flow cytometric analysis of T cells (CD31) cul-tured with allogeneic liver DEC2051B2201CD192 cells demon-strated extensive apoptosis (TUNEL1) (;20–30%) as early as18 h after stimulation, which increased over the next 72 h. BMIL-4 DC or liver GM-CSF DC induced apoptosis in,5–10% ofallogeneic T cells (Fig. 6). Cells were double stained with TUNELand anti-CD4 or anti-CD8 mAbs to determine the subset of T cellsundergoing apoptosis. Liver-derived DEC2051B2201CD192

cells induced similar levels of apoptosis in both CD41 and CD81

T cells (data not shown).

Liver-derived DEC2051B2201CD192 cells induce Trdifferentiation

T cell differentiation following interaction with various subsetsof allogenic DC was determined by measuring cytokine levelsin the supernatants of 2- to 4-day MLR by ELISA. T cellscultured with BM IL-4 DC (mature myeloid DC) secreted typ-ical Th1 cytokines, including IFN-g and IL-2 (Fig. 7A). T cells

FIGURE 6. Identification of apoptotic T cells in MLR. C3H (H-2k) splenic T cells were cultured withg-irradiated B10 (H-2b) BM IL-4 DC, liverGM-CSF DC, or liver-derived DEC2051B2201CD192 cells at S:R ratios of 1:10 for 1–3 days and double stained with TUNEL and anti-CD3. Flowcytometric analysis of gated CD31 cells demonstrates significant apoptosis in T cells stimulated by liver-derived DEC2051B2201CD192 cells occurringas early as day 1 and continuing through day 3. Cells incubated with label solution in the absence of terminal transferase served as controls (openhistograms). Results are representative of three separate experiments.

7047The Journal of Immunology

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

stimulated by liver GM-CSF DC (immature myeloid DC) pro-duced TGF-b, with only low levels of IL-2, IL-4, and IL-10,and no IFN-g, a profile consistent with Th3 differentiation (33).In contrast, T cells were driven by liver-derived DEC2051

B2201CD192 cells to release large amounts of IL-10 andIFN-g, moderate amounts of TGF-b, and very little IL-2 orIL-4. Such a cytokine profile resembles that of Tr1 cells (1, 2,4). Cytokine production by T cells was further confirmed byflow cytometric analysis at a single-cell level. Initially, cellswere double stained to detect H-2k (responder T cell MHC classI) and cytokine. This revealed that the vast majority of cellsproducing IL-10 or IFN-g were T cells (H-2k1; data notshown). Multiple-color staining for IL-10 and IFN-g allowedidentification of a population of cells producing both cytokines.Narrowing the gate to include unusually large cells (gate R2 inFig. 7B), a high proportion (.60%) of gated C3H (H-21) Tcells stimulated by liver-derived DEC2051B2201CD192 cellsreleased both IFN-g and IL-10 (Fig. 7B), a pattern resemblingTr1 cells (1– 4).

Cytokine production by liver DEC2051B2201CD192 cells

Cytokines produced by DC play a critical role in T cell differ-entiation (9, 19). We investigated the cytokine production ofliver DEC2051B2201CD192 cells compared with mature and

immature myeloid DC. CD11c1 BM IL-4 DC and liver GM-CSF DC, as well as liver DEC2051B2201CD192 cells werepurified by flow sorting (99% purity), and cultured for 48 h ina resting state or after activation with LPS. Cytokine levels inthe supernatants were assessed by ELISA. In the resting state,all three types of APC produced low levels of cytokines andNO. LPS stimulation induced cytokine production in all typesof APC, but with a distinctly different pattern for each subset.BM IL-4 DC released large amounts of IL-12, TNF-a, and NO,and moderate amounts of IFN-g. Liver GM-CSF DC respondedto LPS stimulation by markedly increased production of NOand TNF-a, with less pronounced increases in IL-12, IFN-g,and IL-10 production. Thus, upon activation by LPS, the my-eloid DC, in particular mature myeloid DC, released a charac-teristic cytokine pattern capable of inducing Th1 differentiation.In contrast, liver-derived DEC2051B2201CD192 cells se-creted large amounts of IL-10 and IFN-g in response to LPS.TNF-a, IL-12, and NO production were not induced (Fig. 8A).This cytokine pattern (high IL-10 and IFN-g, low IL-12) maybe conducive to Tr1 development. Cytokine mRNA expressionin these cells was consistent with ELISA results. Liver-derivedDEC2051B2201CD192 cells expressed message for the p35subunit of IL-12, but lacked expression of IL-12 p40 (Fig. 8B).Biological function of IL-12 requires the expression of both

FIGURE 7. Cytokine profiles of T cells stimu-lated by various DC.A, Cytokine levels in super-natants from 3-day MLR were assayed by ELISA.C3H (H-2k) splenic T cells were cultured withg-ir-radiated B10 (H-2b) BM IL-4 DC, liver GM-CSFDC, or liver-derived DEC2051B2201CD192 cellsat a S:R ratio of 1:10 for 2–4 days. Data are ex-pressed as pg/ml6 1 SD from triplicate cultures.Liver-derived DEC2051B2201CD192 cells stimu-lated a distinctive cytokine pattern in T cells: highIL-10 and IFN-g, moderate TGF-b, and low IL-2and IL-4. B, Cytometric analysis of single cell in-tracellular cytokine staining. C3H splenic T cellswere cultured withg-irradiated B10 liver-derivedDEC2051B2201CD192 cells at a S:R ratio of 1:10for 3 days. The cells were stained with anti-IL-10FITC and anti-IFN-g PE. Flow cytometric analysisshows that, when gating on the large T cell (H-2k1)population (R2), 50–60% of cells were positive forboth IL-10 and IFN-g. Results are representative ofthree separate experiments.

7048 DEC2051, B2201, CD192 CELLS REGULATE T CELL DIFFERENTIATION

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

subunits (34). Both p35 and p40 mRNA were detected in BMIL-4 DC and inducible in liver GM-CSF DC (Fig. 8B). Theseresults indicate that the signals necessary for T cell differenti-ation can be provided by the APC alone, independent of addi-tional exogenous signals. Mature myeloid DC express cytokinesfavoring Th1 differentiation, whereas liver-derived DEC2051

B2201CD192 cells release cytokines promoting Tr cellpolarization.

Migration of liver-derived DEC2051B2201CD192 cells

DC resident in tissue traffic to draining lymph nodes after Ag pro-cessing or inflammatory stimuli, presumably to present Ag to lym-phocytes. We examined the in vivo migration pattern of DEC2051

B2201CD192 cells derived from liver. A total of 53 105 purifiedB10 liver DEC2051B2201CD192 cells was injected into the foot-pad of allogeneic C3H recipients. Donor-derived cells were iden-tified by immunohistochemistry utilizing mAb specific to donorMHC class II (I-Ab). I-Ab1 cells were visible in draining popliteallymph nodes 1–2 days after injection, but rapidly disappeared afterthat. Subsequently, I-Ab1 cells became detectable in the spleen, locatedpredominantly in T lymphocyte-dependent areas in close proximity toarterioles (Fig. 9). Thus, liver-derived DEC2051B2201CD192 cells ex-hibit a similar homing ability to that described for mature myeloid DC(BM IL-4 DC) and immature myeloid DC (liver GM-CSF DC) (22, 35).

Administration of DEC2051B2201CD192 cells prolongscardiac allograft survival

The ability of liver-derived DEC2051B2201CD192 cells to in-duce T cell apoptosis and promote T cell differentiation consistentwith a Tr phenotype suggests that they may play a role in limitingthe immune response or maintaining tolerance in vivo. This wasassessed in a vascularized cardiac allograft model. A total of 23106 DEC2051B2201CD192 cells propagated from B10 liver NPCwas injected i.v. at various time points before B10 heart transplan-tation into C3H recipients. Mature myeloid DC (BM IL-4 DC),high in costimulatory molecule and MHC expression, and imma-ture myeloid DC (liver GM-CSF DC), deficient in costimulatorymolecule expression, were similarly injected for comparison. Ad-ministration of liver-derived DEC2051B2201CD192 cells signif-icantly prolonged cardiac allograft survival (median survival time(MST) 37 days, compared with MST 10.5 days in nontreated con-trols, p , 0.05) (Fig. 10). The optimal time of administration ofthese cells was 7–10 days before transplantation. Two of six graftsachieved long-term survival (.100 days), with evidence for sys-temic donor-specific tolerance, as exhibited by acceptance of asubsequent donor skin graft. The effects of liver-derivedDEC2051B2201CD192 cells were donor specific, as they failedto prolong survival of BALB/c cardiac allografts. As previously

FIGURE 8. Cytokine profiles of DC with or withoutLPS stimulation.A, B10 BM IL-4 DC (a), liver GM-CSF DC (b) or liver-derived DEC2051B2201CD192

cells (c) were purified by flow cytometry to.99% pu-rity, then cultured with or without the addition of LPS(10 mg/ml, for an additional 48 h of culture). IL-10,IL-12, TNF-a, IFN-g, and NO were measured in culturesupernatants by ELISA (or colorimetric assay based onthe Griess reaction for NO). Results are expressed asmean picograms per milliliter for cytokines, and micro-molar for NO6 SD of triplicate experiments.B, Cyto-kine mRNA expression in DC was determined byRNase protection assay. Results are representative ofthree separate experiments.

7049The Journal of Immunology

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

reported, administration of BM IL-4 DC exacerbated rejection ofcardiac allografts (MST 5 days,p , 0.05 compared with non-treated controls). Liver GM-CSF DC slightly, but not significantly,prolonged allograft survival (Fig. 10).

DiscussionThere appear to be several pathways for development of DC. In allcases, the cells exhibit typical DC morphology, and express dis-tinctive surface molecules involved in Ag uptake (macrophagemannose receptor, DEC205), Ag presentation (MHC I, MHC II),and costimulation (CD40, CD80, CD86) (36). However, the de-velopmental pathways differ in origin of the cells and the resultingfunction of the mature progeny.

The classic myeloid DC develop in response to GM-CSF fromCD341 progenitors into cells identical to epidermal Langerhanscells (37). A second myeloid pathway develops from a CD141

intermediate in response to GM-CSF and IL-4 (37, 38). These cells

share some surface markers with macrophages, and are character-istic of interstitial DC.

Lymphoid DC develop through another pathway. They share aprecursor with T cells and lack myeloid markers (12–15, 20, 39).They may express lymphoid markers such as CD4 or CD8. Ratherthan responding to GM-CSF, they appear to propagate in the pres-ence of IL-3, IL-1, or CD40 ligation. They may express Fas ligandand induce apoptosis in T cells in an Ag-specific fashion (15).Lymphoid DC may play a role in maintaining peripheral tolerance,whereas myeloid DC appear to be important for inducing an im-mune response (9, 11).

The data shown in this study give rise to supplementary evi-dence that cytokines, such as GM-CSF and IL-3, influence thedevelopment of cells toward myeloid or lymphoid lineage. Prop-agation of liver NPC in the presence of GM-CSF results in my-eloid DC that express myeloid Ag CD11c, while cells propagatedin response to IL-3 and CD40 ligation evolve into lymphoid DCthat express DEC205 instead of CD11c, and share a number ofcharacteristics with B cells, including expression of B220 and Iggene rearrangements. However, they lack the B cell marker CD19,and do not express Ig, except for small amounts ofk light chain,on the cell surface. However, these liver-derived DEC2051B2201

CD192 cells exhibit typical DC morphology, and migrate to T cellareas in the spleen and lymph nodes after s.c. injection (40). Theyexpress molecules necessary for Ag presentation and costimula-tion, and activate T cells in an allogeneic MLR. Expression ofB220 on B cells typically occurs only upon maturation; gene re-arrangement can be detected in pre-B cells. Expression of thek-light chain is characteristic of immature B cells. Thus, the cellsdescribed in these experiments probably derive from a commonprecursor with B cells, and may represent a subset of B cells thathas acquired properties consistent with DC (morphology, pheno-type, T cell stimulatory capacity, migratory ability).

The expression of B220 is not limited to B cells. B2201

CD192NK1.11 cells can be obtained from mouse BM, and sub-sequently develop into NK cells following culture in IL-2 (41).However, the DEC2051B2201CD192 cells described in this workcannot be propagated from BM or spleen (data not shown), sug-gesting that either cofactors present in the liver are necessary fortheir development, or that they arise from a different precursor.

Recently, several reports have described DC that share charac-teristics with B cells. A population of CD191 pro-B cells developsinto DC with strong allostimulatory capacity when cultured in IL-1b, IL-3, IL-7, TNF-a, stem cell factor, and Flt-3 ligand (42).Similarly, CD191 cells expressingk- or l-chain, and with DCmorphology can be isolated from human blood mononuclear cells(43). They express the DC marker CD83, and show potent allo-stimulatory activity in MLR. These reports suggest that a subtypeof DC and B cells develops from a common precursor with po-tential to differentiate into either cell type.

A number of reports have described the potential of cells of theB lineage to differentiate into macrophages (44, 45). Even at a latestage of differentiation, pre-B cells expressing surface Ig receptorcomplexes with surrogate L chains can be induced by IL-3 to dif-ferentiate into macrophages with a loss of pre-B cell features. Fur-thermore, IL-3 appears to play an important role in the develop-ment and proliferation of B cells. Although supportive of pro- andpre-B cells in long-term culture, it suppresses early B lymphopoi-esis, and inhibits further differentiation into surface Ig-producing Bcells (46–48). Additional stimulation through CD40 promotesproliferation and differentiation of pre-B cells into immature Bcells (49–51). In contrast, CD40 ligation inhibits the growth anddevelopment of pro-B cells. Thus, the culture conditions in ourexperiments would seem to inhibit B cell development.

FIGURE 10. Administration of liver-derived DEC2051B2201 CD192

cells significantly prolongs cardiac allograft survival in a donor-specificfashion. A total of 23 106 B10 mature myeloid DC (BM IL-4 DC),immature myeloid DC (liver GM-CSF DC), or liver-derivedDEC2051B2201CD192 cells was injected i.v. into C3H (H-2k) recipients7 days before transplantation of a vascularized cardiac graft from B10(H-2b) or BALB/c (H-2d, third-party) donor. Liver-derived DEC2051

B2201CD192 cells significantly prolonged survival of cardiac allograftsfrom B10, but not BALB/c mice. BM IL-4 DC accelerated allograft rejec-tion. n 5 6 in each group.

FIGURE 9. Migration and survival of B10 (H-2b) liver-derivedDEC2051B2201CD192 cells in allogeneic recipients. A total of 23 105

sorted DEC2051B2201CD192 cells was injected into the hind footpad ofC3H (H-2k) mice. Cryostat sections of spleen were stained with donor-specific anti-I-Ab mAb. Photomicrograph of spleen 2 days after injectionshows cells bearing donor I-Ab Ag localized to the white pulp, mainly inthe T cell-dependent region in proximity to the central arteriole (originalmagnification,3400). Inset, Details of cells bearing I-Ab Ag (originalmagnification,31000).

7050 DEC2051, B2201, CD192 CELLS REGULATE T CELL DIFFERENTIATION

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

The general inhibitory effects of IL-3 and CD40 ligation onearly B lymphopoiesis are in contrast to their effects on the devel-opment of myeloid cells. IL-3 synergizes with IL-6 and G-CSF topromote proliferation of myeloid progenitors (48, 52, 53). CD40ligation of CD341 progenitors induces their proliferation and dif-ferentiation into cells bearing many characteristics of DC. Activa-tion of CD40 is one of the most powerful signals for inducing finalmaturation of DC (54–56). Liver-derived DEC2051B2201

CD192 cells may thus derive from a B lymphoid precursor that hasdifferentiated into a cell with characteristics of DC.

The in vivo trafficking of the cells in our experiments is moreconsistent with DC than B cells. Naive B cells are only poorlymigratory. Effector or memory B cells usually traffic to tertiarylymphoid tissues such as found in the skin or intestinal laminapropria (57, 58). In contrast, liver-derived DEC2051B2201

CD192 cells migrate to draining lymph nodes and spleen, wherethey can be found in the T-dependent areas.

It was unexpected that, unlike BM IL-4 DC, phenotypically ma-ture DEC2051B2201CD192 cells induce a low [3H]TdR incor-poration of T cells in MLR, and those low proliferative T cellsproduce high cytokines. This can be explained by the data shownin this study that the T cells stimulated by DEC2051B2201

CD192 cells are undergoing activation. They, however, die of ap-optosis, as inhibition of apoptosis by addition of the caspase in-hibitor peptide z-VAD-fmk restores high thymidine uptake (Fig.5C). In addition, stimulation by DEC2051B2201CD192 cells pro-motes Tr1-like cell differentiation. Tr1 cells are known to producecytokines while having low proliferative capacity (1–4). The ex-tensive apoptosis noted in T cells activated by these cells maycontribute to tolerance induction. The precise molecular eventsinvolved in the process, and whether T cell apoptosis is inducedeither by the DEC2051B2201CD192 cells or by Tr cells, are cur-rent topics of investigation.

The ability of these cells to induce Tr1 differentiation (as defined byT cells with low proliferative capacity, and productive of IFN-g, IL-10, and TGF-b, but no IL-2 or IL-4) raises the possibility that theymay play a role in maintaining peripheral tolerance or limiting im-mune reactivity. Tr1 development was originally described in an invivo mouse model using chronic Ag stimulation (59). Subsequentstudies demonstrated that T lymphocytes stimulated repeatedly in thepresence of IL-10 developed into Tr1 cells, adoptive transfer of whichprotected against inflammatory autoimmune disease in vivo (1). Asimilar cell population was also identified in transgenic mice express-ing a single TCR and its cognate Ag (60). There have been varyingdefinitions of Tr1 cells since they were first described as capable ofsecreting large quantities of IL-10, moderate amounts of TGF-b, andno IL-2, IL-4, and IFN-g (1). A recent paper reported murine Tr1 cellsas a group of T cells that produce high IL-10, moderate TGF-b andIFN-g, and low IL-2 and IL-4 (2). Sologa et al. (4) has defined T cellssecreting both IL-10 and IFN-g as Tr1-like cells. We demonstrate inthis study that the T cells elicited by liver DEC2051B2201CD192

cells have characteristics of Tr1 cells since they have low proliferativecapacity, and produce IL-10, IFN-g, but no IL-2 and IL-4. There is nodirect evidence that the TGF-b detected in MLR supernatants (Fig.7A) is produced by T cells. The low affinity mAb for TGF-b is in-adequate for intracellular staining, as suggested by several manufac-turers. However, it is unlikely that the TGF-b detected in MLR su-pernatants by ELISA (.1000 pg/ml) is produced by DC because theratio of irradiated DC:T cells was only 1:10, with the T cells faroutnumbering the DC. Further substantiation was demonstrated whenRNase protection assay and ELISA revealed no production of TGF-bmRNA or protein by liver DEC2051B2201CD192 cells (data notshown). The exact mechanisms underlying Tr1 induction remain to bedescribed, but IL-10 promotes Tr1 development, and low IL-12 levels

are permissive of non-Th1 differentiation (61, 62). The liver-derivedDEC2051B2201CD192 cells produce IL-10 in the absence of IL-12,and may provide the antigenic signal necessary for Tr1 generationwithout the need for chronicity (i.e., only a single stimulation of Tcells is needed in vitro).

Liver-derived DEC2051B2201CD192 cells exhibit tolerogenicproperties in vivo, as evidenced by their ability to dramaticallyprolong cardiac allograft survival after a single injection. The de-velopment of donor-specific tolerance, as demonstrated by survivalof a subsequent skin graft, suggests that these cells play an activerole in tolerance induction and maintenance in this model. Therelative roles of T cell apoptosis and Tr generation in vivo areunknown. Similar mechanisms may be active in the tolerance as-sociated with liver transplantation exhibited in several animal spe-cies. DEC2051B2201CD192 cells may develop in vivo followingtransplantation or some other inflammatory stimulus. Followingplacement of the allograft, the influx of inflammatory cells asso-ciated with the procedure provides an ample source of IL-3 andCD40 ligand (63–66).

Our results are consistent with the evolving concept of func-tional heterogeneity of DC subsets. Different subsets to date de-scribed have the ability to drive Th1 and Th2 differentiation, orinduce T cell apoptosis in an Ag-dependent manner. The cellsdescribed in this work induce Tr differentiation and apoptosis ofactivated T cells, making them ideal candidates for maintainingperipheral tolerance.

AcknowledgmentsWe thank Dr. Angus W. Thomson for critically reviewing this manuscript;Mamie H. Dong for assistance with ELISA; Allison Logar for assistancewith flow cytometry; and Donna Stolz for assistance with preparation andprocessing of electron microscopy specimens.

References1. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antinenko J. E. de Vries, and

M. G. Roncarolo. 1997. A CD41 T-cell subset inhibits antigen-specific T-cellresponses and prevents colitis.Nature 389:737.

2. Roncarolo, M.-G., M. K. Levings, and C. Traversari. 2001. Differentiation of Tregulatory cells by immature dendritic cells.J. Exp. Med. 193:5.

3. Helmut, J., E. Schmitt, G. Schuler, J. Knop, and A. H. Enk. 2000. Induction ofinterleukin 10-producing, nonproliferating CD41 T cells with regulatory prop-erties by repetitive stimulation with allogeneic immature human dendritic cells.J. Exp. Med. 192:1213.

4. Sologa, J., I. Bellinghausen, and J. Knop. 1999. Do Tr1 cells play a role inimmunotherapy?Int. Arch. Allergy Immunol. 118:210.

5. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, andK. M. Murphy. 1993. Development of TH1 CD41 T cells through IL-12 pro-duced byListeria-induced macrophages.Science 260:547.

6. Schmitt, E., P. Hoehn, T. Germann, and E. Rude. 1994. Differential effects ofinterleukin-12 on the development of naive mouse CD41 T cells.Eur. J. Immu-nol. 24:343.

7. Swain, S. L., A. D. Weinberg, M. English, and G. Huston. 1990. IL-4 directs thedevelopment of Th2-like helper effectors.J. Immunol. 145:3796.

8. Kaplan, M. H., U. Schindler, S. T. Smiley, and M. J. Grusby. 1996. STAT 6 isrequired for mediating responses to IL-4 and for development of Th2 cells.Im-munity 4:313.

9. Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity.Annu. Rev. Immunol. 9:271.

10. Steinman, R. M., S. Turley, and I. Mellman. 2000. The induction of tolerance bydendritic cells that have captured apoptotic cells.J. Exp. Med. 191:411.

11. Steinman, R. M., and K. Inaba. 1999. Myeloid dendritic cells.J. Leukocyte Biol.66:205.

12. Ardavin, C., L. Wu., C. L. Li., and K. Shortman. 1993. Thymic dendritic cells andT cells develop simultaneously in the thymus from a common precursor popu-lation. Nature 362:761.

13. Wu, L., C. L. Li., and K. Shortman. 1996. Thymic dendritic cell precursors:relationship to the T lymphocyte lineage and phenotype of the dendritic cellprogeny.J. Exp. Med. 184:903.

14. Vremec, D., and K. Shortman. 1997. Dendritic cell subtypes in mouse lymphoidorgans: cross-correlation of surface markers, changes with incubation, and dif-ferences among thymus, spleen, and lymph nodes.J. Immunol. 159:565.

15. Suss, G., and K. Shortman. 1996. A subclass of dendritic cells kills CD4 T cellsvia Fas/Fas-ligand-induced apoptosis.J. Exp. Med. 183:1789.

16. Kronin, V., K. Winkel, G. Suss, A. Kelso, W. Heath, J. Kirberg, H. von Boehmer,and K. Shortman. 1996. A subclass of dendritic cells regulates the response ofnaive CD8 T cells by limiting their IL-2 production.J. Immunol. 157:3819.

7051The Journal of Immunology

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

17. Fazekas de St Groth, B. 1998. The evolution of self-tolerance: a new cell arisesto meet the challenge of self-reactivity.Immunol. Today 19:448.

18. Iwasaki, A., and B. L. Kelsall. 1999. Freshly isolated Peyer’s patch, but notspleen, dendritic cells produce interleukin-10 and induce the differentiation of Thelper type 2 cells.J. Exp. Med. 190:229.

19. Rissoan, M.-C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere,R. de Waal Malefyt, and Y.-J. Liu. 1998. Reciprocal control of T helper cell anddendritic cell differentiation.Science 283:1183.

20. Grouard, F., M.-C. Rissoan, L. Filgueira, I. Durand, J. Banchereau, and Y.-J. Liu.1997. The enigmatic plasmacytoid T cells develop into dendritic cells with in-terleukin (IL)-3 and CD40-ligand.J. Exp. Med. 185:1101.

21. Swiggard, W. J., A. Mirza, M. C. Nussenzweig, and R. M. Steinman. 1995.DEC-205, a 205-kDa protein abundant on mouse dendritic cells and thymic ep-ithelium that is detected by the monoclonal antibody NLDC-145: purification,characterization, and N-terminal amino acid sequence.Cell. Immunol. 165:302.

22. Lu, L., J. Woo, A. S. Rao, Y. Li, S. C. Watkins, S. Qian, T. E. Starzl,A. J. Demetris, and A. W. Thomson. 1994. Propagation of dendritic cell progen-itors from normal mouse liver using GM-CSF and their maturational develop-ment in the presence of type-1 collagen.J. Exp. Med. 179:1823.

23. Lu, L., D. McCaslin, T. E. Starzl, and A. W. Thomson. 1995. Mouse bone mar-row-derived dendritic cell progenitors (NLDC 1451, MHC class II1, B7-1dim,B7-22) induce alloantigen-specific hyporesponsiveness in murine T lympho-cytes.Transplantation 60:1539.

24. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu,and R. M. Steinman. 1992. Generation of large numbers of dendritic cells frommouse bone marrow cultures supplemented with granulocyte macrophage colo-ny-stimulating factors. J. Exp. Med. 176:1693.

25. Khanna, A., A. E. Morelli, C. Zhong, L. Lu, and A. W. Thomson. 2000. Effectsof liver-derived dendritic cell progenitors on Th1- and Th2-like cytokine responsein vitro and in vivo.J. Immunol. 164:1346.

26. Lu, L., C. A. Bonham, F. G. Chambers, S. C. Watkins, R. A. Hoffman,R. L. Simmons, and A. W. Thomson. 1996. Induction of nitric oxide synthase inmouse dendritic cells by IFN-g, endotoxin, and interaction with allogeneic Tcells: nitric oxide production is associated with dendritic cell apoptosis.J. Im-munol. 157:3577.

27. Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation byacid guanidinium thiocyanate-phenol chloroform extraction.Anal. Biochem. 162:156.

28. Schlissel, M. S., L. M. Corcoran, and D. Baltimore. 1991. Virus-transformedpre-B cells show ordered activation but not inactivation of immunoglobulin generearrangement an transcription.J. Exp. Med. 173:711.

29. Ono, K., and E. S. Lindsey. 1969. Improved technique of heart transplantation inrats. J. Thorac. Cardiovasc. Surg. 7:225.

30. Drakes, M., L. Lu, V. Subbotin, and A. W. Thomson. 1997. In vivo administra-tion of FLT3 ligand markedly simulates generation of dendritic cell progenitorsfrom mouse liver. J. Immunol. 64:1808.

31. Janeway, C. A., Jr., P. Travers, M. Walport, and J. D. Capra. 1999. The devel-opment of B lymphocytes. InImmunobiology, Vol. 6. Current Biology Publica-tions and Garland Publishing, New York, p. 195.

32. Pronk, G. J., K. Ramer, P. Amiri, and L. T. Williums. 1996. Requirement of anICE-like protease for induction of apoptosis and ceramide generation by REPER.Science 271:808.

33. Inobe, J., A. J. Slavin, Y. Komagata, Y. Chen, L. Liu, and H. L. Weiner. 1998.IL-4 is a differentiation factor for transforming growth factor-b secreting Th3cells and oral administration of IL-4 enhances oral tolerance in experimentalallergic encephalomyelitis.Eur. J. Immunol. 28:2780.

34. Gubler, U., A. O. Chua, D. S. Schoenhaut, C. M. Dwyer, W. McComas,R. Motyka, N. Nabavi, A. G. Wolitzky, P. M. Quinn, and P. C. Familletti. 1991.Coexpression of two distinct genes is required to generate secreted bioactivecytotoxic lymphocyte maturation factor.Proc. Natl. Acad. Sci. USA 88:4143.

35. Thomson, A. W., L. Lu, V. M. Subbotin, Y. Li, S. Qian, A. S. Rao, J. J. Fung,and T. E. Starzl. 1995. In vivo propagation and homing of liver-derived dendriticcell progenitors to lymphoid tissue of allogeneic recipients: implications for theestablishment and maintenance of donor cell chimerism following liver trans-plantation.Transplantation 59:544.

36. Steinman, R. M. 1999. Dendritic cells. InFundamental Immunology, Vol. 16.W. E. Paul, ed. Lippincott-Raven Publishers, Philadelphia, p. 547.

37. Caux, C., B. Vanbervliet, C. Massacrier, C. Dezutter-Dambuyant,B. de Saint-Vis, C. Jacquet, K. Yoneda, S. Imamura, D. Schmitt, andJ. Banchereau. 1996. CD341 hematopoietic progenitors from human cord blooddifferentiate along two independent dendritic cell pathways in response to GM-CSF1TNF a. J. Exp. Med. 184:695.

38. Szabolcs, P., D. Avigan, S. Gezelter, D. H. Ciocon, M. A. Moore,R. M. Steinman, and J. W. Young. 1996. Dendritic cells and macrophages canmature independently from a human bone marrow-derived, post-colony-formingunit intermediate.Blood 87:4520.

39. Vremec, D., M. Zorbas, R. Scollay, D. J. Saunders, C. F. Ardavin, L. Wu, andK. Shortman. 1992. The surface phenotype of dendritic cells purified from mousethymus and spleen: investigation of the CD8 expression by a subpopulation ofdendritic cells.J. Exp. Med. 176:47.

40. Austyn, J. M., J. W. Kupiec-Weglinski, D. F. Hankins, and P. J. Morris. 1988.Migration patterns of dendritic cells in the mouse: homing to T cell-dependentareas of spleen, and binding within marginal zone.J. Exp. Med. 167:646.

41. Rolink, A., E. ten Boekel, F. Melchers, D. T. Fearon, I. Krop, and J. Anderson.1996. A subpopulation of B2201 cells in murine bone marrow does not expressCD19 and contain natural killer cell progenitors.J. Exp. Med. 183:187.

42. Bjorck, P., and P. W. Kincade. 1998. CD191 pro-B cells can give rise to dendriticcells in vitro.J. Immunol. 161:5795.

43. Zhong, R.-K., A. D. Donnenberg, H.-F. Zhang, S. Watkins, J.-H. Zhou, andE. D. Ball. 1999. Human blood dendritic cell-like B cells isolated by the 5G9monoclonal antibody reactive with novel 220-kDa antigen.J. Immunol. 163:1354.

44. Cumano, A., C. J. Paige, N. N. Iscove, and G. Brady. 1992. Bipotential precursorsof B cells and macrophages in murine fetal liver.Nature 356:612.

45. Martin, M., A. Strasser, N. Baumgarth, F. M. Cicuttini, K. Welch, E. Salvaris,and A. W. Boyd. 1993. A novel cellular model (SPGM 1) of switching betweenthe pre-B cell and myelomonocytic lineages.J. Immunol. 150:4395.

46. Ball, T. C., F. Hirayama, and M. Ogawa. 1996. Modulation of early B lympho-poiesis by interleukin-3.Exp. Hematol. 24:1225.

47. Winkler, T. H., F. Melchers, and A. G. Rolink. 1995. Interleukin-3 and interleu-kin-7 are alternative growth factors for the same B-cell precursors in the mouse.Blood 85:2045.

48. Matsunaga, T., F. Hirayama, Y. Yonemura, R. Murray, and M. Ogawa. 1998.Negative regulation by interleukin-3 (IL-3) of mouse early B-cell progenitors andstem cells in culture: transduction of the negative signals bybc andbIL-3 pro-teins of IL-3 receptor and absence of negative regulation by granulocyte-mac-rophage colony-stimulating factor.Blood 92:901.

49. Larson, A. W., and T. W. LeBien. 1994. Cross-linking CD40 on human B cellprecursors inhibits or enhances growth depending on the stage of developmentand the IL costimulus.J. Immunol. 153:584.

50. Kindler, V., and R. H. Zubler. 1997. Memory, but not naive, peripheral blood Blymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimu-lation with IL-4 and the differentiation factors IL-2, IL-10, and IL-3.J. Immunol.159:2085.

51. Van Kooten, C., and J. Banchereau. 2000. CD40-CD40 ligand.J. Leukocyte Biol.67:2.

52. Ikebuchi, K., G. G. Wong, S. C. Clark, J. N. Ihle, Y. Hirai, and M. Ogawa. 1987.Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipo-tential hemopoietic progenitors.Proc. Natl. Acad. Sci. USA 84:9035.

53. Ikebuchi, K., S. C. Clark, J. N. Ihle, L. M. Souza, and M. Ogawa. 1988. Gran-ulocyte colony-stimulating factor enhances interleukin 3-dependent proliferationof multipotential hemopoietic progenitors.Proc. Natl. Acad. Sci. USA 85:3445.

54. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control ofimmunity. Nature 392:245.

55. Mueller, C. G., M. C. Rissoan, B. Salina, S. Ait-Yahia, O. Ravel, J. M. Bridon,F. Briere, S. Lebecque, and Y. J. Liu. 1997. Polymerase chain reaction selects anovel disintegrin proteinase from CD40-activated germinal center dendritic cells.J. Exp. Med. 186:655.

56. Flores-Romo, L., P. Bjorck, V. Duvert, C. van Kooten, S. Saeland, andJ. Banchereau. 1997. CD40 ligation on human cord blood CD341 hematopoieticprogenitors induces their proliferation and differentiation into functional dendriticcells.J. Exp. Med. 185:341.

57. Picker, L. J., and E. C. Butcher. 1992. Physiological and molecular mechanismsof lymphocyte homing.Annu. Rev. Immunol. 10:561.

58. Butcher, E. C., and L. J. Picker. 1996. Lymphocyte homing and homeostasis.Science 272:60.

59. Sundstedt, A., I. Hoiden, A. Rosendahl, T. Kalland, N. Van Rooijen, andM. Dohlsten. 1997. Immunoregulatory role of IL-10 during superantigen-inducedhyporesponsiveness in vivo.J. Immunol. 158:180.

60. Buer, J., A. Lanoue, A. Franzke, C. Garcia, H. von Boehmer, and A. Sarukhan. 1998.Interleukin-10 secretion and impaired effector function of major histocompatibilitycomplex class II-restricted T cells anergized in vivo.J. Exp. Med. 187:177.

61. Seder, R. A., R. Gazzinelli, A. Sher, and W. E. Paul. 1993. Interleukin-12 actsdirectly on CD41 T cells to enhance priming for interferong production anddiminishes interleukin-4 inhibition of such priming.Proc. Natl. Acad. Sci. USA90:10188.

62. Schmitt, E., P. Hoehn, T. Germann, and E. Rude. 1994. Differential effects ofinterleukin-12 on the development of naive mouse CD41 T cells.Eur. J. Immu-nol. 24:343.

63. Durie, F. H., T. M. Foy, S. R. Masters, J. D. Laman, and R. J. Noelle. 1994. Therole of CD40 in the regulation of humoral and cell-mediated immunity.Immunol.Today 15:406.

64. Lane, P. J., and T. Brocker. 1999. Developmental regulation of dendritic cellfunction.Curr. Opin. Immunol. 11:308.

65. Nishinakamura, R., A. Miyajima, P. J. Mee, V. L. J. Tybulewicz, and R. Murray.1996. Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions.Blood 88:2458.

66. Alvarez-Silva, M., and R. Borojevic. 1996. GM-CSF and IL-3 activities in schis-tosomal liver granulomas are controlled by stroma-associated heparan sulfateproteoglycans.J. Leukocyte Biol. 59:435.

7052 DEC2051, B2201, CD192 CELLS REGULATE T CELL DIFFERENTIATION

by guest on July 28, 2018http://w

ww

.jimm

unol.org/D

ownloaded from