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The Journal of Immunology, 2001, 166: 7042-7052.
Copyright © 2001 by The American Association of Immunologists

Liver-Derived DEC205+B220+CD19- Dendritic Cells Regulate T Cell Responses1

Lina Lu2,*, C. Andrew Bonham*, Xiaoyan Liang*, Zongyou Chen*, Wei Li*, Liangfu Wang*, Simon C. Watkins{dagger}, Michael A. Nalesnik{ddagger}, Mark S. Schlissel§, Anthony J. Demestris{ddagger}, John J. Fung* and Shiguang Qian*

* Thomas E. Starzl Transplantation Institute and Department of Surgery, {dagger} Department of Cell Biology and Physiology, and {ddagger} Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213; and § Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205brightB220+CD11c-CD19-, and negative for T (CD3, CD4, CD8{alpha}), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to 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 DEC205+B220+D19- cells release both IL-10 and IFN-{gamma}, small amounts of TGF-{beta}, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-{gamma}, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell differentiation is crucial to the outcome of an immune response. Early in the process of activation, T cells are committed to develop into one of several functionally distinct subsets, including Th1, Th2, and the recently described T regulatory (Tr)3 cells. Tr cells may play a critical role in the generation and maintenance of tolerance.

Several varieties of Tr cells have been described, each with unique, albeit somewhat nebulous characteristics. Thus, a number of definitions of Tr cells exist in the literature (1, 2, 3). Type 1 Tr (Tr1) cells are a subset characterized by their unique profile of cytokine production. Tr1 cells produce high levels of IL-10, moderate amounts of TGF-{beta} and IFN-{gamma}, but no IL-4 or IL-2. They exert immunoregulatory or suppressive effects (1, 2, 3, 4).

T cell differentiation is regulated by the local microenvironment. Hence, the property of Ags encountered by the T cell, and the expression of costimulatory molecules and cytokines by APCs drive T cell differentiation. In vitro, IL-12 drives Th1 (5, 6), whereas IL-4 promotes Th2 differentiation (7, 8). Similarly, the generation of Tr1 cells is driven by IL-10 (1). The stimulus controlling T cell differentiation during an in vivo immune response is less clear.

Dendritic cells (DC) are uniquely suited for activation of naive T cells (9). Recent data suggest that different DC subsets provide T cells with selective signals that guide either Th1 or Th2 differentiation. In mice, DC have been classified into myeloid and lymphoid subsets according to their phenotype and their development from distinct precursors (10, 11, 12, 13, 14). These subsets of DC share a number of distinct properties, including dendritic morphology, the ability to migrate, and expression of a range of molecules required for activation of naive T cells. However, they differ in their regulation of the immune response. Thus, myeloid DC usually initiate immune responses, and typically induce Th1 differentiation. In contrast, the so-called lymphoid DC propagated in response to IL-3, while capable of activating lymphocytes, may also limit T cell proliferation by inducing Fas-mediated apoptosis and inhibiting cytokine production (15, 16, 17, 18). Analogous to mice, humans may also contain two DC types developed from distinct precursors. DC1, propagated in response to GM-CSF from peripheral blood monocytes, produce high levels of IL-12 and induce Th1 differentiation. On the other hand, DC2 propagated from blood or tonsil plasmacytoid T cells in response to IL-3, drive Th2 differentiation (19, 20). Furthermore, repetitive stimulation with allogeneic immature DC induces IL-10-producing, nonproliferating T cells with regulatory properties (3).

In studies designed to assess liver-derived DC and their function in vitro and in vivo, we have identified a novel cell population propagated from normal mouse liver nonparenchymal cells (NPC) in response to IL-3 and anti-CD40 mAb. These cells exhibit DC morphology and express the DC marker DEC205 (21). They also bear the B220 Ag, a marker of cell activation typically associated with B cells, but do not express the B cell Ag CD19. They activate T cells, with subsequent induction of T cell apoptosis. A smaller proportion of T cells is stimulated to release IFN-{gamma}, IL-10, and TGF-{beta}, cytokines resembling a Tr1 cell phenotype. The propagated cells exhibit Ig gene rearrangements consistent with developing B cells, but lack surface expression of Ig. After injection into allogeneic recipients, they migrate to spleen and dramatically prolong a subsequent cardiac allograft.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male C57BL/10 (B10; H-2b), C3H (H-2k), and BALB/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used at 8–12 wk of age. Animals were maintained in the specific pathogen-free facility of the University of Pittsburgh Medical Center (Pittsburgh, PA) and provided with Purina rodent chow (Ralston Purina, St. Louis, MO) and tap water ad libitum.

Isolation of NPC from liver

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

Propagation of DEC205+, B220+, CD19- cells from livers

Liver NPC were depleted of T, B, NK, granular cells, and macrophages by complement-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, 2 x 106 lineage-negative cells were cultured in 2 ml of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with antibiotics and 10% (v/v) FCS (referred to subsequently as complete medium), and mouse rIL-3 (10 ng/ml; BioSource International, Camarillo, CA), plus anti-CD40 mAb (2 ng/ml; PharMingen) in flat-bottom 24-well culture plates for 5–7 days. Nonadherent cells released from clusters were harvested for further characterization. For comparative purposes, mature myeloid DC propagated from bone marrow (BM) in GM-CSF plus IL-4 (referred to subsequently as BM IL-4 DC) and immature myeloid DC propagated from liver NPC in GM-CSF alone (referred to subsequently as liver GM-CSF DC), as described elsewhere (22, 23), were used. Briefly, BM cells or liver NPC were cultured in 24-well plates (2 x 106/well) in complete medium containing both mouse rGM-CSF (4 ng/ml) and rIL-4 (1000 U/ml) (both from Schering-Plough, Kenilworth, NJ) or GM-CSF alone for 5–7 days. The selection and purification procedures were similar to those reported 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 an Epics Elite flow cytometer (Coulter, Hialeah, FL). FITC- or PE-conjugated mAbs were obtained from PharMingen, except for anti-DEC-205 mAb (generously provided by R. M. Steinman, The Rockefeller University, New York, NY). For intracellular cytokine detection, cells were incubated in brefeldin A (10 µg/ml; Sigma) for 5 h, then washed with 1% saponin/1% FCS/PBS, as described previously (25). Double staining was performed using FITC- or PE-conjugated anti-H-2Kb, anti-IL-10, or anti-IFN-{gamma} mAbs. Cells were then washed with 1% FCS/PBS and resuspended in 1% formaldehyde before analysis. Appropriate isotype- and species-matched irrelevant mAbs were used as controls.

Detection of apoptosis

T cells were stained with PE-conjugated anti-CD3{epsilon}, anti-CD4, or anti-CD8{alpha} mAb, and DNA strand breaks were identified by TUNEL. Following surface CD3, CD4, or CD8 staining, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate. TUNEL reaction mixture of the Cell Death Detection kit (Roche Diagnostics, Indianapolis, IN) was then added according to the manufacturer’s instructions. Cells incubated with label solution in the absence of terminal transferase were used as negative controls. Quantitative analysis was performed by flow cytometry, with 5000 events acquired from each sample. For identification of apoptotic cells in cytospin preparations, T cells activated by DC were processed for immunocytochemical detection of incorporated biotin-dUTP by peroxidase-labeled avidin, followed by an enzyme reaction using aminoethylcarbazole as the substrate, as described elsewhere (26).

Mixed leukocyte reaction

To determine the allostimulatory capacity of DC, one-way MLR were performed with {gamma}-irradiated (20 Gy) DC or spleen cells from B10 (allogeneic) or C3H (syngeneic) mice as stimulators and nylon wool-purified C3H spleen T cells (2 x 105) as responders. Cultures (200 µl) were established in triplicate in 96-well round-bottom microculture plates and maintained in complete medium in 5% CO2 in air at 37°C for 3–4 days. [3H]TdR (1 µCi/well) was added for the final 18 h of culture, and incorporation of [3H]TdR into DNA was assessed by liquid scintillation counting in an automated counter. Results are expressed as mean cpm ± 1 SD. In apoptosis inhibition experiments, a common caspase inhibitor peptide, benzyloxycarbonyl-Val Ala-Asp-fluoromethyl ketone (zVAD-fmk; Alexis, San Diego, CA), was added (100 µM) at the beginning of the MLR culture. DMSO served as a control.

Cytokine and NO quantitation

IL-2, IFN-{gamma}, IL-4, IL-10, IL-12, TNF-{alpha}, and TGF-{beta} levels in supernatants of MLR or DC cultures were quantitated using ELISA kits (BioSource International), with sensitivity limits of 20–25 pg/ml, as described (25). A standard curve using recombinant cytokine was generated for each assay. NO levels were determined by the colorimetric Griess reaction that detects the 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 multiprobe RNase protection assay system (PharMingen) following the manufacturer’s instruction. Briefly, 5 µg of total RNA was hybridized to 32P-labeled RNA probes overnight at 56°C, followed by treatment with RNase for 45 min at 30°C. The murine L32 and GAPDH riboprobes were used as controls. Protected fragments were submitted to electrophoresis through a 7 M urea/5% polyacrylamide gel and then exposed to Kodak X-OMAT film (Kodak, Rochester, NY) for 72 h.

DNA PCR assay for Ig rearrangement

DNA was prepared for PCR by lysing cells in 200 µl 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 60 µg/ml proteinase K), incubating them at 55°C for 1 h, and then inactivating the protease by heating to 95°C for 10 min. DNA at a concentration of 5000 genomes/µl was used for PCR. PCR (50 µl) were performed as described previously (28). Thirty cycles of amplification were performed, after which one-fifth of each reaction was analyzed on a 1.4% agarose gel in Tris-borate buffer. The gel was blot transferred to a nylon membrane and probed with 32P-labeled DNA from the appropriate Ig C region. Primer sequences are as published elsewhere (28). Germline alleles were detected using a primer (Mu0) 322 nt 5' to JH1. DH R and L primers are oligonucleotide mixtures degenerate at two and three positions, respectively, and homologous to all members of the Dfl16 and Dsp2 D gene families. D to J rearrangements were detected as amplified fragments of ~1033, ~716, or ~333 nt depending on whether JH1, JH2, or JH3 was rearranged. To detect V to DJ rearrangements, a mixture of three different degenerate oligonucleotides homologous to conserved framework region 3 sequences of three VH gene families (VH7183, VH558, and VHQ52) and the J3 primer was used. This results in amplified VDJ rearrangements of ~1058, ~741, or ~358 nt. PCR products were detected by hybridization with appropriate Ig gene probes. Both DJ and VDJ rearrangements result in loss of Mu0 sequence and its amplification product. V to DJ rearrangement events result in loss of all of the DH L primer target sequences and amplified DJ fragments.

In vivo migration

Cells propagated from B10 mice were injected s.c. (5 x 105 cells in 50 µl) into a hind footpad of normal allogeneic C3H recipients. Animals were sacrificed in groups of three at days 1, 2, 3, and 7 after injection. The draining popliteal lymph node, thymus, and spleen were removed, embedded in Tissue-Tek OCT compound (Miles, Elkart, IN), and frozen at -80°C. Cryostat sections (4 µm) were air dried at room temperature overnight for further processing.

Immunohistochemistry

B10 MHC class II+ cells were identified in cryostat sections or cytospin preparations using biotinylated mouse IgG2a anti-mouse I-Ab (PharMingen) in an avidin-biotin-alkaline phosphatase complex (ABC) staining procedure. Isotype- and species-matched irrelevant mAb were used as control. Donor MHC class II+ (I-Ab+) cells were counted in 100 high-power fields, and the data were expressed as number of I-Ab+ cells per high-power field.

Heterotopic vascularized heart transplantation

Surgical procedures were performed under methoxyflurane (Medical Development, Springvale, Australia) inhalation anesthesia. Cardiac anastomoses to the abdominal aorta and inferior vena cava were performed as described previously (29). The function of the donor heart was monitored daily by abdominal palpation. Rejection was defined as total cessation of contraction, which was confirmed by histological examination.

Statistical analyses

Graft survival times between groups of transplanted animals were compared using the Mann-Whitney U test. A p <0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of DEC205+B220+CD19- cells from liver NPC

NPC were isolated from livers of B10 mice. Approximately 7–8 x 106 cells were obtained from each liver, with <5% hepatocyte contamination. An Ab mixture and complement were used to deplete CD4+, CD8{alpha}+, CD14+, CD19+, NK1.1+, and Gr-1+ cells. Cells were then cultured in complete medium containing IL-3 for 3–4 days, and clusters of proliferating cells were noted (Fig. 1GoA). Addition of anti-CD40 mAb induced the formation of long dendritic process on these cells. Following 3–4 additional days in culture, the cells detached from the adherent clusters. By 6–8 days, ~3 x 106 such cells were obtained from each mouse liver.



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  		FIGURE 1. Cells propagated from normal B10 (H-2b) liver NPC with IL-3 and anti-CD40 mAb. A, Cell clusters at 48 h of culture. B, Immunocytochemical staining for MHC class II (I-Ab) expression of cells cultured for 6 days, displaying typical DC morphology with long, thin, and beaded processes.

 
Morphologically, the cells displayed characteristics of DC, including irregular-shaped eccentric nuclei, a paucity of prominent cytoplasmic granules, and extended dendrites (Fig. 1GoB). Transmission electron microscopy further delineated the delicate cytoplasmic processes, an abundance of mitochondria, and the absence of electron-dense granules (Fig. 2GoA). The typical dendritic veils or pseudopodia were observed under scanning electron microscopy (Fig. 2Go, B and C).



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  		FIGURE 2. Ultrastructure of liver NPC-derived cells propagated with IL-3 and anti-CD40 mAb. A, Transmission electron micrographs of cells cultured for 6 days demonstrate numerous, extensive cytoplasmic processes, irregularly shaped nuclei, numerous mitochondria, and a paucity of paracrystalline cytoplasmic granules. B and C, Scanning electron micrographs of cells showing veils (3-day (B) and 5-day (C) culture). Bars, 1 µm.

 
Immunophenotypic analysis demonstrated high expression of CD45, MHC class I, MHC class II, costimulatory molecules (CD40, CD80, and CD86), and the lymphoid DC marker DEC-205 (Fig. 3GoA). The myeloid DC marker CD11c was absent. Additionally, the cells did not express Ags associated with myeloid cells (CD13, CD11b, or CD14), T cells (CD3{epsilon}, CD4, and CD8{alpha}), or NK cells (NK1.1). Of interest, the cells expressed B220, an Ag typically present on B cells, but lacked the B cell-restricted molecule CD19 (Fig. 3GoB).



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  		FIGURE 3. A, Flow cytometric analysis of cell surface Ag expression on liver-derived DEC205+B220+ CD19- cells propagated with IL-3 and anti-CD40 mAb (filled histograms) compared with liver GM-CSF DC (open histograms). Appropriate Ig isotype controls are shown as dotted profiles. B, Liver DEC205+B220+ CD19- cells were double stained with PE anti-DEC205 and FITC anti-B220 or FITC anti-CD19. Data are representative of three separate experiments.

 
By contrast, liver GM-CSF DC expressed the myeloid lineage molecules CD11b, CD13, and CD14, as well as the myeloid DC marker CD11c (Fig. 3GoA), as described (22). They exhibited only low levels of DEC-205. MHC and costimulatory molecule expression were low (Fig. 3GoA), 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 associated with Ag presention can be propagated from precursors present in liver NPC in response to different cytokines (IL-3/CD40 ligand vs GM-CSF). BM IL-4 DC show a mature myeloid DC phenotype (data not shown), as reported (23).

Ig gene rearrangement and expression in liver-derived DEC205+B220+CD19- cells

The expression of B220 by cells derived from liver NPC in response to IL-3 and CD40 ligation raises the possibility that they derive from B cells. Rearrangement of Ig genes occurs relatively early in B cell development, and are detectable in all but the earliest precursors. DNA was isolated from purified liver-derived DEC205+B220+CD19-cells (sorted by flow cytometry to achieve purity >99%), and analyzed by PCR for Ig gene rearrangements. D to JH, V to DJH, and V to J{kappa} gene rearrangements were identified in the cells (Fig. 4GoA). A similar pattern was noted in splenocytes rich in mature B cells. In contrast, myeloid DC had only Ig heavy chain DJ rearrangements. This is not surprising, as many T cells and other myeloid lineage cells have similar rearrangements. However, the myeloid DC lack VDJ and VJ{kappa} alleles (Fig. 4GoA).



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  		FIGURE 4. A, DNA PCR analysis for Ig gene rearrangements in liver-derived DEC205+B220+CD19- cells. DNA was isolated from CD11c+ BM-IL-4 DC (lane 1), or liver DEC205+B220+CD19- cells (lanes 2 and 3, representing samples from two experiments) purified by flow cytometry. Lane 4, DNA ladder. Thymus and spleen cells are represented in lanes 5 and 6. A RAG-deficient pro-B cell line 63-12 is shown in lane 7. Lane 8, Cell-free negative control. D to JH, V to DJH, and V to J{kappa} gene rearrangements were identified in DEC205+B220+CD19- cells. B, Flow cytometric analysis of Ig expression on liver DEC205+B220+CD19- cells. Cells were double stained with anti-B220 PE or DEC205 PE and anti-CD19, anti-IgG, anti-IgM, anti-Ig{kappa}, or anti-Ig{lambda} FITC mAb, showing these DEC205+B220+CD19- cells do not express IgG and IgM, but express low Ig{kappa}. The data are representative of three separate experiments.

 
To determine expression of Ig proteins, the DEC205+B220+ CD19- cells were stained with mAbs specific to mouse IgG, IgM, Ig{kappa}, or Ig{lambda}. The cells lacked expression of IgG, IgM, and Ig{lambda}. A small proportion of cells expressed low levels of Ig{kappa} {lambda} (Fig. 4GoB). Expression of {kappa} light chain is normally found in immature B cells in conjunction with a µ-heavy chain to form IgM (31). These data suggest that DEC205+B220+CD19- cells have developed from B cell precursors (pro-B or pre-B), but have arrested or diverged at some point before becoming immature B cells.

Allostimulatory capacity of liver-derived DEC205+B220+ CD19- cells

The allostimulatory capacity of DEC205+, B220+, CD19- cells derived from B10 liver NPC was determined in a one-way MLR. Mature myeloid DC (BM IL-4 DC) stimulated vigorous allogenic T cell proliferation, whereas liver-derived DEC205+B220+CD19- cells induced very little T cell proliferation, as determined by thymidine uptake. The allostimulatory capacity was similar to that seen with immature myeloid DC propagated from liver in response to GM-CSF (liver GM-CSF DC) (Fig. 5GoA) (22). Low T cell proliferation after stimulation by liver GM-CSF DC was expected due to low expression of MHC and costimulatory molecules (Fig. 3GoA) (22). However, the DEC205+B220+CD19- cells expressed high levels of these molecules (Fig. 3GoA) and would be expected to stimulate a brisk T cell response. Direct inspection of the T cells over the course of the MLR revealed evidence of T cell death. After 2 days in culture, similar levels of T blasts developed in re-sponse to BM IL-4 DC and liver-derived DEC205+B220+ CD19- cells. Few blasts were observed in T cells responding to liver GM-CSF DC. However, T cells stimulated by liver-derived DEC205+B220+CD19- cells rapidly died, as determined by in situ TUNEL staining. After 3 days in culture, a large number of apoptotic cells mixed with large blast cells were visible in T cells stimulated by liver DEC205+B220+CD19- cells (Fig. 5GoB). In contrast, few T cells stimulated by BM IL-4 DC or liver GM-CSF DC exhibited evidence of cell death. The low thymidine uptake by T cells stimulated by liver-derived DEC205+B220+CD19- cells, despite the appearance of T cell blasts, must therefore be due to rapid apoptosis of activated T cells. Indeed, inhibition of apoptosis by addition of the caspase inhibitor peptide zVAD-fmk (32) restored T cell proliferation induced by liver-derived DEC205+ B220+CD19- cells (Fig. 5GoC).



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  		FIGURE 5. A, [3H]TdR uptake by T cells in MLR. C3H (H-2k) splenic T cells were cultured with {gamma}-irradiated B10 (H-2b) spleen cells, mature myeloid DC (BM IL-4 DC), immature myeloid DC (liver GM-CSF DC), or liver-derived DEC205+ B220+CD19- cells for 3 days at various S:R ratios. B, Cytocentrifuge preparations of cells from 3-day culture were stained by in situ nick-end labeling (TUNEL). T cells cultured with liver DEC205+B220+CD19- cells at T:DC ratio of 10:1 demonstrated higher levels of apoptosis than T cells cultured with BM IL-4 DC or liver GM-CSF DC. Original magnification, x100. C, Allostimulatory activity of liver-derived DEC205+B220+CD19- cells was restored by addition of the common caspase inhibitor zVAD-fmk (100 µM) at the beginning of 3-day MLR. Results are expressed as mean cpm ± SD of triplicate cultures and are representative of three experiments.

 
Liver-derived DEC205+B220+CD19- cells induce T cell apoptosis

To confirm that T cells stimulated by allogenic liver DEC205+ B220+CD19- cells undergo apoptosis, C3H splenic T cells cultured with various DC subtypes from B10 donors for 1–3 days were stained by TUNEL and anti-CD3, anti-CD4, or anti-CD8 mAbs. Two-color flow cytometric analysis of T cells (CD3+) cultured with allogeneic liver DEC205+B220+CD19- cells demonstrated extensive apoptosis (TUNEL+) (~20–30%) as early as 18 h after stimulation, which increased over the next 72 h. BM IL-4 DC or liver GM-CSF DC induced apoptosis in <5–10% of allogeneic T cells (Fig. 6Go). Cells were double stained with TUNEL and anti-CD4 or anti-CD8 mAbs to determine the subset of T cells undergoing apoptosis. Liver-derived DEC205+B220+CD19- cells induced similar levels of apoptosis in both CD4+ and CD8+ T cells (data not shown).



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  		FIGURE 6. Identification of apoptotic T cells in MLR. C3H (H-2k) splenic T cells were cultured with {gamma}-irradiated B10 (H-2b) BM IL-4 DC, liver GM-CSF DC, or liver-derived DEC205+B220+CD19- cells at S:R ratios of 1:10 for 1–3 days and double stained with TUNEL and anti-CD3. Flow cytometric analysis of gated CD3+ cells demonstrates significant apoptosis in T cells stimulated by liver-derived DEC205+B220+CD19- cells occurring as early as day 1 and continuing through day 3. Cells incubated with label solution in the absence of terminal transferase served as controls (open histograms). Results are representative of three separate experiments.

 
Liver-derived DEC205+B220+CD19- cells induce Tr differentiation

T cell differentiation following interaction with various subsets of allogenic DC was determined by measuring cytokine levels in the supernatants of 2- to 4-day MLR by ELISA. T cells cultured with BM IL-4 DC (mature myeloid DC) secreted typical Th1 cytokines, including IFN-{gamma} and IL-2 (Fig. 7GoA). T cells stimulated by liver GM-CSF DC (immature myeloid DC) produced TGF-{beta}, with only low levels of IL-2, IL-4, and IL-10, and no IFN-{gamma}, a profile consistent with Th3 differentiation (33). In contrast, T cells were driven by liver-derived DEC205+ B220+CD19- cells to release large amounts of IL-10 and IFN-{gamma}, moderate amounts of TGF-{beta}, and very little IL-2 or IL-4. Such a cytokine profile resembles that of Tr1 cells (1, 2, 4). Cytokine production by T cells was further confirmed by flow cytometric analysis at a single-cell level. Initially, cells were double stained to detect H-2k (responder T cell MHC class I) and cytokine. This revealed that the vast majority of cells producing IL-10 or IFN-{gamma} were T cells (H-2k+; data not shown). Multiple-color staining for IL-10 and IFN-{gamma} allowed identification of a population of cells producing both cytokines. Narrowing the gate to include unusually large cells (gate R2 in Fig. 7GoB), a high proportion (>60%) of gated C3H (H-2+) T cells stimulated by liver-derived DEC205+B220+CD19- cells released both IFN-{gamma} and IL-10 (Fig. 7GoB), a pattern resembling Tr1 cells (1, 2, 3, 4).



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  		FIGURE 7. Cytokine profiles of T cells stimulated by various DC. A, Cytokine levels in supernatants from 3-day MLR were assayed by ELISA. C3H (H-2k) splenic T cells were cultured with {gamma}-irradiated B10 (H-2b) BM IL-4 DC, liver GM-CSF DC, or liver-derived DEC205+B220+CD19- cells at a S:R ratio of 1:10 for 2–4 days. Data are expressed as pg/ml ± 1 SD from triplicate cultures. Liver-derived DEC205+B220+CD19- cells stimulated a distinctive cytokine pattern in T cells: high IL-10 and IFN-{gamma}, moderate TGF-{beta}, and low IL-2 and IL-4. B, Cytometric analysis of single cell intracellular cytokine staining. C3H splenic T cells were cultured with {gamma}-irradiated B10 liver-derived DEC205+B220+CD19- cells at a S:R ratio of 1:10 for 3 days. The cells were stained with anti-IL-10 FITC and anti-IFN-{gamma} PE. Flow cytometric analysis shows that, when gating on the large T cell (H-2k+) population (R2), 50–60% of cells were positive for both IL-10 and IFN-{gamma}. Results are representative of three separate experiments.

 
Cytokine production by liver DEC205+B220+CD19- cells

Cytokines produced by DC play a critical role in T cell differentiation (9, 19). We investigated the cytokine production of liver DEC205+B220+CD19- cells compared with mature and immature myeloid DC. CD11c+ BM IL-4 DC and liver GM-CSF DC, as well as liver DEC205+B220+CD19- cells were purified by flow sorting (99% purity), and cultured for 48 h in a resting state or after activation with LPS. Cytokine levels in the supernatants were assessed by ELISA. In the resting state, all three types of APC produced low levels of cytokines and NO. LPS stimulation induced cytokine production in all types of APC, but with a distinctly different pattern for each subset. BM IL-4 DC released large amounts of IL-12, TNF-{alpha}, and NO, and moderate amounts of IFN-{gamma}. Liver GM-CSF DC responded to LPS stimulation by markedly increased production of NO and TNF-{alpha}, with less pronounced increases in IL-12, IFN-{gamma}, and IL-10 production. Thus, upon activation by LPS, the myeloid DC, in particular mature myeloid DC, released a characteristic cytokine pattern capable of inducing Th1 differentiation. In contrast, liver-derived DEC205+B220+CD19- cells secreted large amounts of IL-10 and IFN-{gamma} in response to LPS. TNF-{alpha}, IL-12, and NO production were not induced (Fig. 8GoA). This cytokine pattern (high IL-10 and IFN-{gamma}, low IL-12) may be conducive to Tr1 development. Cytokine mRNA expression in these cells was consistent with ELISA results. Liver-derived DEC205+B220+CD19- cells expressed message for the p35 subunit of IL-12, but lacked expression of IL-12 p40 (Fig. 8GoB). Biological function of IL-12 requires the expression of both subunits (34). Both p35 and p40 mRNA were detected in BM IL-4 DC and inducible in liver GM-CSF DC (Fig. 8GoB). These results indicate that the signals necessary for T cell differentiation can be provided by the APC alone, independent of additional exogenous signals. Mature myeloid DC express cytokines favoring Th1 differentiation, whereas liver-derived DEC205+ B220+CD19- cells release cytokines promoting Tr cell polarization.



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  		FIGURE 8. Cytokine profiles of DC with or without LPS stimulation. A, B10 BM IL-4 DC (a), liver GM-CSF DC (b) or liver-derived DEC205+B220+CD19- cells (c) were purified by flow cytometry to >99% purity, then cultured with or without the addition of LPS (10 µg/ml, for an additional 48 h of culture). IL-10, IL-12, TNF-{alpha}, IFN-{gamma}, and NO were measured in culture supernatants by ELISA (or colorimetric assay based on the Griess reaction for NO). Results are expressed as mean picograms per milliliter for cytokines, and micromolar for NO ± SD of triplicate experiments. B, Cytokine mRNA expression in DC was determined by RNase protection assay. Results are representative of three separate experiments.

 
Migration of liver-derived DEC205+B220+CD19- cells

DC resident in tissue traffic to draining lymph nodes after Ag processing or inflammatory stimuli, presumably to present Ag to lymphocytes. We examined the in vivo migration pattern of DEC205+ B220+CD19- cells derived from liver. A total of 5 x 105 purified B10 liver DEC205+B220+CD19- cells was injected into the footpad of allogeneic C3H recipients. Donor-derived cells were identified by immunohistochemistry utilizing mAb specific to donor MHC class II (I-Ab). I-Ab+ cells were visible in draining popliteal lymph nodes 1–2 days after injection, but rapidly disappeared after that. Subsequently, I-Ab+ cells became detectable in the spleen, located predominantly in T lymphocyte-dependent areas in close proximity to arterioles (Fig. 9Go). Thus, liver-derived DEC205+B220+CD19- cells exhibit 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).



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  		FIGURE 9. Migration and survival of B10 (H-2b) liver-derived DEC205+B220+CD19- cells in allogeneic recipients. A total of 2 x 105 sorted DEC205+B220+CD19- cells was injected into the hind footpad of C3H (H-2k) mice. Cryostat sections of spleen were stained with donor-specific anti-I-Ab mAb. Photomicrograph of spleen 2 days after injection shows cells bearing donor I-Ab Ag localized to the white pulp, mainly in the T cell-dependent region in proximity to the central arteriole (original magnification, x400). Inset, Details of cells bearing I-Ab Ag (original magnification, x1000).

 
Administration of DEC205+B220+CD19- cells prolongs cardiac allograft survival

The ability of liver-derived DEC205+B220+CD19- cells to induce T cell apoptosis and promote T cell differentiation consistent with a Tr phenotype suggests that they may play a role in limiting the immune response or maintaining tolerance in vivo. This was assessed in a vascularized cardiac allograft model. A total of 2 x 106 DEC205+B220+CD19- cells propagated from B10 liver NPC was injected i.v. at various time points before B10 heart transplantation into C3H recipients. Mature myeloid DC (BM IL-4 DC), high in costimulatory molecule and MHC expression, and immature myeloid DC (liver GM-CSF DC), deficient in costimulatory molecule expression, were similarly injected for comparison. Administration of liver-derived DEC205+B220+CD19- cells significantly prolonged cardiac allograft survival (median survival time (MST) 37 days, compared with MST 10.5 days in nontreated controls, p < 0.05) (Fig. 10Go). The optimal time of administration of these cells was 7–10 days before transplantation. Two of six grafts achieved long-term survival (>100 days), with evidence for systemic donor-specific tolerance, as exhibited by acceptance of a subsequent donor skin graft. The effects of liver-derived DEC205+B220+CD19- cells were donor specific, as they failed to prolong survival of BALB/c cardiac allografts. As previously reported, administration of BM IL-4 DC exacerbated rejection of cardiac allografts (MST 5 days, p < 0.05 compared with nontreated controls). Liver GM-CSF DC slightly, but not significantly, prolonged allograft survival (Fig. 10Go).



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  		FIGURE 10. Administration of liver-derived DEC205+B220+ CD19- cells significantly prolongs cardiac allograft survival in a donor-specific fashion. A total of 2 x 106 B10 mature myeloid DC (BM IL-4 DC), immature myeloid DC (liver GM-CSF DC), or liver-derived DEC205+B220+CD19- cells was injected i.v. into C3H (H-2k) recipients 7 days before transplantation of a vascularized cardiac graft from B10 (H-2b) or BALB/c (H-2d, third-party) donor. Liver-derived DEC205+ B220+CD19- cells significantly prolonged survival of cardiac allografts from B10, but not BALB/c mice. BM IL-4 DC accelerated allograft rejection. n = 6 in each group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There appear to be several pathways for development of DC. In all cases, the cells exhibit typical DC morphology, and express distinctive surface molecules involved in Ag uptake (macrophage mannose receptor, DEC205), Ag presentation (MHC I, MHC II), and costimulation (CD40, CD80, CD86) (36). However, the developmental pathways differ in origin of the cells and the resulting function of the mature progeny.

The classic myeloid DC develop in response to GM-CSF from CD34+ progenitors into cells identical to epidermal Langerhans cells (37). A second myeloid pathway develops from a CD14+ intermediate in response to GM-CSF and IL-4 (37, 38). These cells share some surface markers with macrophages, and are characteristic of interstitial DC.

Lymphoid DC develop through another pathway. They share a precursor with T cells and lack myeloid markers (12, 13, 14, 15, 20, 39). They may express lymphoid markers such as CD4 or CD8. Rather than responding to GM-CSF, they appear to propagate in the presence of IL-3, IL-1, or CD40 ligation. They may express Fas ligand and 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 immune response (9, 11).

The data shown in this study give rise to supplementary evidence that cytokines, such as GM-CSF and IL-3, influence the development of cells toward myeloid or lymphoid lineage. Propagation of liver NPC in the presence of GM-CSF results in myeloid DC that express myeloid Ag CD11c, while cells propagated in response to IL-3 and CD40 ligation evolve into lymphoid DC that express DEC205 instead of CD11c, and share a number of characteristics with B cells, including expression of B220 and Ig gene rearrangements. However, they lack the B cell marker CD19, and do not express Ig, except for small amounts of {kappa} light chain, on the cell surface. However, these liver-derived DEC205+B220+ CD19- cells exhibit typical DC morphology, and migrate to T cell areas in the spleen and lymph nodes after s.c. injection (40). They express molecules necessary for Ag presentation and costimulation, and activate T cells in an allogeneic MLR. Expression of B220 on B cells typically occurs only upon maturation; gene rearrangement can be detected in pre-B cells. Expression of the {kappa}-light chain is characteristic of immature B cells. Thus, the cells described in these experiments probably derive from a common precursor with B cells, and may represent a subset of B cells that has acquired properties consistent with DC (morphology, phenotype, T cell stimulatory capacity, migratory ability).

The expression of B220 is not limited to B cells. B220+ CD19-NK1.1+ cells can be obtained from mouse BM, and subsequently develop into NK cells following culture in IL-2 (41). However, the DEC205+B220+CD19- cells described in this work cannot be propagated from BM or spleen (data not shown), suggesting that either cofactors present in the liver are necessary for their development, or that they arise from a different precursor.

Recently, several reports have described DC that share characteristics with B cells. A population of CD19+ pro-B cells develops into DC with strong allostimulatory capacity when cultured in IL-1{beta}, IL-3, IL-7, TNF-{alpha}, stem cell factor, and Flt-3 ligand (42). Similarly, CD19+ cells expressing {kappa}- or {lambda}-chain, and with DC morphology can be isolated from human blood mononuclear cells (43). They express the DC marker CD83, and show potent allostimulatory activity in MLR. These reports suggest that a subtype of DC and B cells develops from a common precursor with potential to differentiate into either cell type.

A number of reports have described the potential of cells of the B lineage to differentiate into macrophages (44, 45). Even at a late stage of differentiation, pre-B cells expressing surface Ig receptor complexes with surrogate L chains can be induced by IL-3 to differentiate into macrophages with a loss of pre-B cell features. Furthermore, IL-3 appears to play an important role in the development and proliferation of B cells. Although supportive of pro- and pre-B cells in long-term culture, it suppresses early B lymphopoiesis, and inhibits further differentiation into surface Ig-producing B cells (46, 47, 48). Additional stimulation through CD40 promotes proliferation and differentiation of pre-B cells into immature B cells (49, 50, 51). In contrast, CD40 ligation inhibits the growth and development of pro-B cells. Thus, the culture conditions in our experiments would seem to inhibit B cell development.

The general inhibitory effects of IL-3 and CD40 ligation on early B lymphopoiesis are in contrast to their effects on the development of myeloid cells. IL-3 synergizes with IL-6 and G-CSF to promote proliferation of myeloid progenitors (48, 52, 53). CD40 ligation of CD34+ progenitors induces their proliferation and differentiation into cells bearing many characteristics of DC. Activation of CD40 is one of the most powerful signals for inducing final maturation of DC (54, 55, 56). Liver-derived DEC205+B220+ CD19- cells may thus derive from a B lymphoid precursor that has differentiated into a cell with characteristics of DC.

The in vivo trafficking of the cells in our experiments is more consistent with DC than B cells. Naive B cells are only poorly migratory. Effector or memory B cells usually traffic to tertiary lymphoid tissues such as found in the skin or intestinal lamina propria (57, 58). In contrast, liver-derived DEC205+B220+ CD19- cells migrate to draining lymph nodes and spleen, where they can be found in the T-dependent areas.

It was unexpected that, unlike BM IL-4 DC, phenotypically mature DEC205+B220+CD19- cells induce a low [3H]TdR incorporation of T cells in MLR, and those low proliferative T cells produce high cytokines. This can be explained by the data shown in this study that the T cells stimulated by DEC205+B220+ CD19- cells are undergoing activation. They, however, die of apoptosis, as inhibition of apoptosis by addition of the caspase inhibitor peptide z-VAD-fmk restores high thymidine uptake (Fig. 5GoC). In addition, stimulation by DEC205+B220+CD19- cells promotes Tr1-like cell differentiation. Tr1 cells are known to produce cytokines while having low proliferative capacity (1, 2, 3, 4). The extensive apoptosis noted in T cells activated by these cells may contribute to tolerance induction. The precise molecular events involved in the process, and whether T cell apoptosis is induced either by the DEC205+B220+CD19- cells or by Tr cells, are current topics of investigation.

The ability of these cells to induce Tr1 differentiation (as defined by T cells with low proliferative capacity, and productive of IFN-{gamma}, IL-10, and TGF-{beta}, but no IL-2 or IL-4) raises the possibility that they may play a role in maintaining peripheral tolerance or limiting immune reactivity. Tr1 development was originally described in an in vivo mouse model using chronic Ag stimulation (59). Subsequent studies demonstrated that T lymphocytes stimulated repeatedly in the presence of IL-10 developed into Tr1 cells, adoptive transfer of which protected against inflammatory autoimmune disease in vivo (1). A similar cell population was also identified in transgenic mice expressing a single TCR and its cognate Ag (60). There have been varying definitions of Tr1 cells since they were first described as capable of secreting large quantities of IL-10, moderate amounts of TGF-{beta}, and no IL-2, IL-4, and IFN-{gamma} (1). A recent paper reported murine Tr1 cells as a group of T cells that produce high IL-10, moderate TGF-{beta} and IFN-{gamma}, and low IL-2 and IL-4 (2). Sologa et al. (4) has defined T cells secreting both IL-10 and IFN-{gamma} as Tr1-like cells. We demonstrate in this study that the T cells elicited by liver DEC205+B220+CD19- cells have characteristics of Tr1 cells since they have low proliferative capacity, and produce IL-10, IFN-{gamma}, but no IL-2 and IL-4. There is no direct evidence that the TGF-{beta} detected in MLR supernatants (Fig. 7GoA) is produced by T cells. The low affinity mAb for TGF-{beta} is inadequate for intracellular staining, as suggested by several manufacturers. However, it is unlikely that the TGF-{beta} detected in MLR supernatants by ELISA (>1000 pg/ml) is produced by DC because the ratio of irradiated DC:T cells was only 1:10, with the T cells far outnumbering the DC. Further substantiation was demonstrated when RNase protection assay and ELISA revealed no production of TGF-{beta} mRNA or protein by liver DEC205+B220+CD19- cells (data not shown). The exact mechanisms underlying Tr1 induction remain to be described, but IL-10 promotes Tr1 development, and low IL-12 levels are permissive of non-Th1 differentiation (61, 62). The liver-derived DEC205+B220+CD19- cells produce IL-10 in the absence of IL-12, and may provide the antigenic signal necessary for Tr1 generation without the need for chronicity (i.e., only a single stimulation of T cells is needed in vitro).

Liver-derived DEC205+B220+CD19- cells exhibit tolerogenic properties in vivo, as evidenced by their ability to dramatically prolong cardiac allograft survival after a single injection. The development of donor-specific tolerance, as demonstrated by survival of a subsequent skin graft, suggests that these cells play an active role in tolerance induction and maintenance in this model. The relative roles of T cell apoptosis and Tr generation in vivo are unknown. Similar mechanisms may be active in the tolerance associated with liver transplantation exhibited in several animal species. DEC205+B220+CD19- cells may develop in vivo following transplantation or some other inflammatory stimulus. Following placement of the allograft, the influx of inflammatory cells associated with the procedure provides an ample source of IL-3 and CD40 ligand (63, 64, 65, 66).

Our results are consistent with the evolving concept of functional heterogeneity of DC subsets. Different subsets to date described have the ability to drive Th1 and Th2 differentiation, or induce T cell apoptosis in an Ag-dependent manner. The cells described in this work induce Tr differentiation and apoptosis of activated T cells, making them ideal candidates for maintaining peripheral tolerance.


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


   Footnotes
 
1 This study was supported by National Institutes of Health Grant DK 29961 and Juvenile Diabetes Foundation International Grant P1893135. Back

2 Address correspondence and reprint requests to Dr. Lina Lu, Thomas E. Starzl Transplantation Institute and Department of Surgery, University of Pittsburgh Medical Center, E1554 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: lul{at}msx.upmc.edu Back

3 Abbreviations used in this paper: Tr, T regulatory; BM, bone marrow; DC, dendritic cell; MST, median survival time; NPC, nonparenchymal cell; S:R, stimulator:responder; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Back

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


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