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(Journal of Leukocyte Biology. 2000;68:633-640.)
© 2000 by Society for Leukocyte Biology

Tacrolimus (FK506) treatment of CD34+ hematopoietic progenitor cells promote the development of dendritic cells that drive CD4+ T cells toward Th2 responses

Kanako Shimizu*,{dagger}, Shin-ichiro Fujii{dagger},{ddagger}, Koji Fujimoto{dagger}, Keisei Kawa§, Akira Yamada* and Fumio Kawano{dagger}

* Department of Immunology, Kurume University School of Medicine, Fukuoka, Japan;
{dagger} The Center for Bone Marrow Transplantation and Immunotherapy, Institute for Clinical Research, Kumamoto National Hospital, Kumamoto, Japan;
{ddagger} Department of Surgery, Biological Therapeutic Program, University of Pittsburgh School of Medicine, and University of Pittsburgh Cancer Institute, Pennsylvania; and
§ Department of Pediatrics, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan

Correspondence: Dr. Shin-ichiro Fujii, Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Ave., New York, NY 10021-6399. E-mail: fujii1018{at}aol.com


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ABSTRACT
 
The macrolide lactone, tacrolimus (FK506), is utilized in bone marrow transplantation (BMT) to prevent graft-versus-host disease (GVHD). In the current study, we evaluated the ability of FK506 to modify the function of dendritic cells (DCs) derived from CD34+ hematopoietic progenitor cells (HPCs). Comparable to DCs obtained in the absence of FK506, DCs cultured in the presence of FK506 (FK-DCs) had higher expression of CD1a+ and formed a greater number of DC colonies. Despite the same expression of costimulatory molecules, FK-DCs displayed a reduced capacity to stimulate an allogeneic T cell response, and showed significantly lower interleukin (IL)-12 production. While normal DCs pulsed with the exogenous antigen, keyhole limpet hemocyanin (KLH) induced specific Th1-like interferon-{gamma}(IFN-{gamma}) producing CD4+ T cell line, FK-DCs induced Th2-like interleukin-4 (IL-4) producing CD4+ T cell line. These data demonstrate the ability of FK506 to induce Th2-promoting function in developing DCs.

Key Words: hematopoietic progenitor cell • dendritic cell • tacrolimus (FK506) • Th2


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INTRODUCTION
 
Dendritic cells (DCs) are potent activators of naive T cells, critical for the initiation of primary specific immune responses [1 , 2 ]. Because spleen- and skin-derived DCs, as well as DCs matured in vitro, all have the capacity to produce interleukin (IL)-12, it is thought that these cells preferentially induce Th1 immune responses [3 , 4 ]. Indeed, Th1 cells are required to generate protective cell-mediated inflammatory responses against invading intracellular microorganisms [5 , 6 ]. It is likely, however, that the nature of this Th1 response is not due solely to the intrinsic properties of the DCs, but is also highly influenced by the biological activities of the antigen and/or microenvironmental factors. For example, IL-12 production by DCs may be induced by exogenous interferon-{gamma} (IFN-{gamma}) derived from activated bystander memory T cells, thus promoting Th1 immune responses [7 ]. Th1 cells may also play a pathogenic role in certain autoimmune diseases, as well as in acute graft-versus-host disease (GVHD) [8 ]. GVHD continues to be a major problem in allogeneic bone marrow transplantation (allo-BMT) and markedly limits the efficacy of this important procedure [9 10 11 ]. Progress in understanding the immunological basis of organ rejection and GVHD has led to attempts to modify the host response to alloantigens [9 , 10 ]. In particular, the Th1/Th2 cell ratio has been shown to be essential in the treatment of GVHD in patients receiving allo-BMT [9 , 12 ].

Some immunosuppressive agents, such as prostaglandin E2 (PGE2), IL-10, corticosteroid (CS), and cyclosporin A have been shown to regulate DCs by enhancing their macrophage-like characteristics [13 14 15 16 17 18 19 20 ]. Although FK506 has been shown to have a direct effect on CD4+ T cells by reducing IL-2 release as well as somewhat influencing the activity of either B cells or macrophages [21 ], direct effects of FK506 on DCs have not been thoroughly investigated. Previous in vivo studies showed that administration of FK506 and flt3-ligand (FL) to mice transplanted with allogeneic bone marrow inhibited anti-donor cytotoxic effects, despite the presence of proliferating DCs [22 , 23 ]. In addition, topical application of FK506 to epidermal cells during primary contact hypersensitivity responses was found to suppress the levels of mRNA encoding the cytokines, IL-1{alpha}, IL-1ß, TNF-{alpha}, granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-2 (MIP-2), and the costimulatory molecules, CD54 and CD80 [24 ]. We therefore assayed the ability of FK506 to alter the differentiation of DCs from hematopoietic progenitor cells (HPCs) and analyzed the ability of DCs obtained in the presence or absence of FK506 to induce either Th1 or Th2 responses.


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MATERIALS AND METHODS
 
Isolation of HPCs
After obtaining informed consent, peripheral blood progenitor cells (PBPCs) that were mobilized with 10 µg/kg recombinant human G-CSF (rhG-CSF; filgastim, Kirin Brewery, Tokyo, Japan) for 3 consecutive days, were obtained from 5 healthy volunteers. Mononuclear cells were isolated from PBPCs by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient separation. Mononuclear cells obtained from the interface were washed with serum-free medium and resuspended in RPMI (GIBCO-BRL, Grand Island, NY) containing 10% fetal calf serum (FCS; GIBCO-BRL). Monocytes were depleted by adherence to plastic culture dishes for 90 min at 37°C. Nonadherent cells were washed and resuspended in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA Fraction V; Sigma Chemical, St. Louis, MO). CD34+ HPCs were positively purified with M-450 Dynabeads coated with anti-CD34 monoclonal antibody (mAb; Dynal, Oslo, Norway) by incubating 4 x 107 beads with 2.5 to 5 x 107 cells for 60 min on ice. Rosetted cells were separated with a magnet, and the beads were detached from the cells with DETACHaBEADs CD34 (Dynal) according to the manufacturer’s instructions. Each preparation contained 87 ± 5% pure viable CD34+ cells [25 , 26 ].

To further purify CD34+ cells as DC progenitors, one million immunomagnetically selected CD34+ cells were incubated for 30 min at 4°C with 20 µL fluorescein isothiocyanate (FITC)-conjugated mAb to CD34 (anti-HPCA-2-FITC, IgG1, Becton Dickinson, San Jose, CA). Mouse IgG1 (FITC+/RPE+/RPE-Cy5+; DAKO A/S, Glostrup, Denmark) was used as the isotype control. The cells were washed twice, resuspended in PBS containing 0.1% BSA, and kept at 4°C before cell sorting on a FACS Vantage (Becton Dickinson). Each preparation contained more than 99% pure viable CD34+ cells.

Generation of DCs and FK-DCs from CD34+ HPCs
CD34+ HPCs were resuspended in RPMI 1640 containing 100 U/mL penicillin, 100 µg/mL kanamycin, 2 mM L-glutamine, and 10% FCS, hereafter designated as complete medium. To develop DCs (thereafter, normal DCs), the culture medium was further supplemented with 100 ng/mL GM-CSF (Kirin Brewery), 2.5 ng/mL tumor necrosis factor {alpha} (TNF-{alpha}; Genzyme, Cambridge, MA), 20 ng/mL stem cell factor (SCF; GIBCO-BRL), and 100 ng/mL FL (PeproTechEC, London, UK) for 2 weeks [27 ]. To develop DCs in the presence of FK506 (FK-DCs), FK506 (Fujisawa Pharmaceutical, Osaka, Japan) was added throughout the culture starting on day 0. The cells were transferred to 12-well culture plates (Nunc, Roskilde, Denmark) at a final concentration of 105 cells/mL and incubated at 37°C in a humidified atmosphere with 5% CO2. Cultures were split every 4–5 days.

Allogeneic mixed leukocyte reaction (Allo-MLR)
T cells were obtained from heparinized blood of unrelated healthy volunteers by density gradient centrifugation and removal of nylon wool-adherent cells. These cells (5 x 104 cells/100 µL) were cultured together with 30-Gy irradiated stimulator cells (up to 2 x 104 cells/100 µL), FK-DCs, normal DCs, PBMCs, or CD14+ monocytes/macrophages from the other subjects, were cultured for 6 days in 96-well U-bottomed culture microplates (Nunc). CD14+ monocytes/macrophages were isolated using CD14 MicroBeads (Miltenyi Biotec, Auburn, CA). For the final 16 h of culture, 1 µCi [3H]thymidine (Amersham, Buckingham, UK) was added to each well.

Methylcellulose culture of CD34+ HPCs in colony-forming assays
To determine the number of DC colony-forming units (CFU-DCs), CD34+ cells were cultured in semisolid medium [27 28 29 ]. Five hundred sorted CD34+ cells were plated in triplicate in 1 mL Iscove’s modified Dulbecco’s medium (IMDM) containing 1.1% methylcellulose, 30% FCS, 10-5 M 2-mercaptoethanol (Sigma), 0.2 mM bovine hemin (Sigma), 100 ng/mL GM-CSF, 2.5 ng/mL TNF-{alpha}, 20 ng/mL SCF, and 100 ng/mL FL, with or without 10-6 M FK506. After 14 days of culture at 37°C in a fully humidified 5% CO2 atmosphere, colonies were defined as aggregates of >50 cells. To confirm that the colonies comprised DC, individual aggregates were examined by microscopy. The cells were plated subsequently on glass slides, and assessed by the indirect immunoalkaline phosphatase method using anti-CD1a mAb and an alkaline phosphatase substrate kit I (Vector Red; VectorLaboratories, Burlingame, CA) [26 ].

Flow cytometry analysis
Cells were incubated with FITC- or PE-conjugated mouse mAbs against CD1a, CD54 (Immunotech, Marseille, France), HLA-DR, CD14 (Ortho Diagnostics, Raritan, NJ), CD40, CD80, and CD86 (PharMingen), or fluorochrome-labeled isotype controls (DAKO), and analyzed on a CYTORON ABSOLUTE flow cytometer (Ortho) using ImmunoCount II software (Ortho).

Cytokine production by normal DCs and FK-DCs
For the analysis of cytokine production by each DC, FK506 was removed after 14 days of culture. The FK-DCs or normal DCs (5 x 105 cells) were cultured in 1 mL culture medium alone or with 0.1 µg/mL lipopolysaccharide (LPS; Difco, Detroit, MI), or 1 µg/mL soluble recombinant CD40L (rCD40L; provided by Immunex Research and Development, Seattle, WA) with/without 1000 IU/mL IFN-{gamma} (R & D Systems, Minneapolis, MN). Each culture supernatant was removed after 72 h, and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) in 96-well microtiter plates (Nunc), using human IL-10 and IL-12p40 ELISA testing kits (R & D Systems) and IL-12p70 ELISA testing kits (Endogen, Minneapolis, MN), (sensitivity limit 3.9, 15, and 3 pg/mL for IL-10, IL-12p40, and IL-12p70, respectively).

Assay for phagocytic activity
FluoSpheres,carboxylate-modified microspheres (Molecular Probes, Eugene, OR), are fluorescein latex beads (diameter; 1.0 µm) used for phagocytosis assay [26 ]. They were added at a concentration of 0.03% (vol/vol) to culture medium on culture day 5, and followed by that after culture for a further 9 days, the total cells were harvested and subjected to flow cytometric analysis.

Establishment of antigen-specific CD4+ T cell lines using KLH-pulsed DCs and KLH-pulsed FK-DCs, and anergy tests by challenging KLH-pulsed DCs
Normal DCs or FK-DCs (1 x 105 cells/mL) pulsed with KLH (50 µg/mL; Calbiochem, Bad Soden, Germany) from day 5 to day 14 were irradiated and cocultured with autologous CD4+ T cells (1 x 105 cells/mL), that had been positively selected with anti-CD4 mAb Dynabeads M-450 and CD4/CD8 DETACHaBEADs, in the presence of IL-2 (100 U/mL; Shionogi Pharm., Osaka, Japan). Subsequently, irradiated autologous PBMCs were added as feeder cells every week for 4 weeks. On day 28, the CD4+ T cell lines (>90% CD4+CD45RO+ cells ) were established.

To test for anergy, each CD4+ T cell line (5 x 104/100 µL) was cocultured with 50-Gy irradiated, KLH-pulsed DCs (3 x 104/100 µL) for 3 days, and pulsed for the final 16 h with [3H]thymidine (1 µCi/well). Cells were harvested and counted as described for the allo-MLR.

Intracellular cytokine staining analysis
IFN-{gamma} and IL-4 in the cytoplasm of CD4+ T cell lines were measured by flow cytometry, as described [30 ]. Briefly, the CD4+ T cells were stimulated by phorbol 12-myristate 13-acetate (PMA) (Sigma) and ionomycin (Sigma) with Brefeldin A (Sigma) for 4 h and continuously treated with FACS lysing and permeabilization solutions (Becton Dickinson). The cells were incubated subsequently with FITC-anti-IFN-{gamma} (Becton Dickinson) and PE-anti-IL-4 (Becton Dickinson) in 0.1% BSA/PBS; FITC-mouse IgG2a and PE-mouse IgG1 (Becton Dickinson) were used as controls. The percentage of cells positive for IFN-{gamma} and IL-4 were counted and evaluated by flow cytometry using a FACScan (Becton Dickinson).

Statistical analysis
[3H]thymidine incorporation in FK-DCs and normal DCs was compared using Student’s paired t test. Data are from representative experiments. Experiments were performed three or four times.


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RESULTS
 
Generation, cell recovery, and cloning efficiency of DCs from CD34+ HPCs in the presence or absence of FK506
We generated DCs by incubating CD34+ HPCs in the presence of the growth factors: GM-CSF, TNF-{alpha}, SCF, and FL. FK506 was added to the culture medium to generate FK-DCs. The attempted concentration of FK506 in our experiments was determined by the previous studies of the interaction between other cells (monocyte, B cell, T cell) and FK506, or the concentration of FK506 in plasma and whole blood for clinical uses [21 , 31 32 33 ]. We compared FK-DCs generated using various concentrations of FK506 from 10-10 to 10-5 M for their ability to stimulate allo-MLR. Less than 10-8 M was not effective, and more than 10-5 M showed some cytotoxicity of DCs based on the fact that, although DCs could proliferate, more than 30% decrease in cell number was demonstrated compared with a control group (data not shown). For the above reasons, 10-6 M FK506 was selected in our study (Fig. 1A ). We utilized 10-6 M FK506 in all of our subsequent experiments. We characterized two populations, normal DCs and FK-DCs, by comparing with CD14+ monocytes/macrophages. As shown in Figure 1B , FK-DCs as well as normal DCs showed stronger allo-MLR activities than CD14+ monocytes/macrophages. It suggests that FK-DCs should be different from macrophage lineage.



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  		Figure 1. (A) Evaluation of the effects of various concentration of FK506 on CD34+ HPC-derived DCs. Stimulations of mixed leukocyte reactions by FK-DCs (FK506; 10-7 M to 10-5 M), normal DCs and PBMCs were shown. On day14, DCs generated from CD34+ cells in the presence or absence of FK506 were irradiated and cultured for 6 days with allogeneic lymphocytes and labeled with [3H]-thymidine for the final 16 h of culture. (B) FK-DCs as well as normal DCs apparently differed from CD14+ cells. Alloantigen-presenting capacity of DCs or FK-DCs on culture day14 were compared with CD14+ monocytes/macrophages using the same method as above. Experiments were performed three times. Each bar represents the mean ± SD of 3 determinations.

In our system, there were no statistically significant differences in the cell yield of both the normal DCs group and the FK-DCs group on day 14 (Table 1 ). When we assayed the clonogenic efficiency of granulocyte-macrophage (GM) and DC precursors within the CD34+ cell fractions, we found that the mean colony-forming efficiency of the GM subfraction was approximately equal in normal DCs and FK-DCs (Table 2 ). In contrast, the clonogenic efficiency in the DC subfractions of FK-DCs was higher than that in the respective fraction of normal DCs using the classification by Reid [28 ] (Table 2) . The number of type 1 DC colonies, defined as a pure DC population consisting of more than 50 diffuse cells with long cytoplasmic processes (Fig. 2A B ) was significantly higher in the FK-DC than in the normal DC populations (Table 2) . Type 2 DC colonies, which are thought to consist of mixed populations of DCs + GMs [28 ], were found to consist of dispersed DCs, together with an aggregate of round granular cells with a regular contour (granulocytes and macrophages; Fig. 2C and 2D ), and were more abundant in the cells grown in the presence of FK506 than in its absence (Table 2) . As shown by immunocytochemistry, both type 1 and type 2 DC colonies verified CD1a+ cells belonging to the DC lineage (Fig. 2B and 2D) . These results suggest that the mobilized CD34+ cells contain progenitors capable of differentiating into DCs, and that the frequency and proliferative capacity of these cells are enhanced by FK506.


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  		Table 1. Cell Number After Stimulation by Cytokine With/Without FK506 (on Day 14)


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  		Table 2. Cloning Efficiency of CD34+ HPCs



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  		Figure 2. Light microscopic analysis of normal DC colonies and FK-DC colonies (A, B) a type 1 DC colony, regarded as a pure DC population, consisting of a diffuse cluster of 5–50 cells with long cytoplasmic processes. (C, D) a type 2 DC colony, regarded as a mixed colony of DCs and macrophages, consisting of a cluster of type 1 cells, together with various sized round granular cells with a regular contour of granulocytes and macrophages. (B, D) both cells were picked up from each colony and were assessed by the immunostaining for CD1a. Experiments were performed three times.

Phenotypic analysis of FK-DCs comparable to those of normal DCs
After culturing CD34+ HPCs for 4–5 days in the presence or absence of FK506, we observed primarily immature DCs with short projections. After a further 10 days of culture, the number of CD1a+ DCs that co-expressed CD14+ was higher in the FK-DCs than in the normal DCs culture (Fig. 3 ). FK-DCs also expressed the costimulatory molecules, CD54, CD80, CD86, and CD40, as well as HLA-DR comparable to those of normal DCs (Fig. 3) .



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  		Figure 3. Expression of cell-surface markers by DCs and FK-DCs on day14 DCs and FK-DCs were incubated with mAb to CD1a, HLA-DR, CD80, CD86, CD54, CD40, and CD14, and assayed by flow cytometry. Both groups were also analyzed by two-color flow cytometry (HLA-DR and CD40, CD1a and CD40, CD1a and CD14).

A weak stimulatory capacity of allo-MLR in FK-DCs
Normal DCs have been shown to be strong stimulators of MHC-mismatched peripheral blood lymphocytes [26 ]. As shown in Figure 1 , we found that although FK-DCs were more potent stimulators of allogeneic lymphocytes than freshly isolated PBMCs, they were less potent than normal DCs. For further purification of CD34+ HPCs, we sorted out using FACS Vantage following the positive selection by magnetic beads. FK-DCs generated from sorted CD34+ HPCs showed apparently fewer effects than normal DCs. A 1:1 mixture of normal DCs and FK-DCs, however, was found to suppress the function of the normal DCs (Fig. 4 ).



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  		Figure 4. Demonstration of reduced stimulation of MLR by FK-DCs comparable to normal DCs, and suppression of normal DCs function by FK-DCs Normal DCs and FK-DCs were generated from the further sorted CD34+ HPCs using a FACS Vantage in this experiment for eliminating other cells. On day 14, allo-stimulatory responses were assayed using normal DCs and FK-DCs. Allo lymphocytes were cultured with DCs, FK-DCs, a 1: 1 mixture of DCs + FK-DCs, or allo-PBMCs, as described in the legend to Figure 1 . Each point represents the mean ± SD of three measurements. Data are representative of three experiments.

Differences in cytokine production by normal DCs and FK-DCs
When we assayed IL-12 (p40 and p70) and IL-10 production in FK-DCs and normal DCs in response to LPS or rCD40L with/without IFN-{gamma}, we found that IL-12 (p40 and p70) production was significantly lower in FK-DCs than in normal DCs. Stimulation by rCD40L and/or IFN-{gamma} enhanced the differences of production of IL-12p70 between two populations (Fig. 5 ). By contrast, there was no significant difference in IL-10 production between the two cell populations (Fig. 5) .



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  		Figure 5. IL-12 (p70 and p40) and IL-10 production by normal DCs or FK-DCs. For these experiments, 5 x 105 normal DCs or FK-DCs were cultured for 72 h with/without LPS, rCD40L, rCD40L + IFN-{gamma}. Experiments were performed four times. *P < 0.05 compared to control by Student’s t test.

Phagocytic activity of FK-DCs similar to normal DCs
We compared the total phagocytic cell population between DCs and FK-DCs from culture day 5 to day 14. The percentage of phagocytic cells was approximately equal in the normal DC and FK-DC cultures (Fig. 6A ). When the gated DC subpopulations in the two cultures were compared, 34.4 ± 1.3% of the normal DC and 31.2 ± 0.8% of the FK-DC cells were phagocytic. Therefore, no differences in phagocytic activity were shown between them.



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  		Figure 6. (A) phagocytic activity of DCs and FK-DCs. Beginning on culture day 5, DCs (1) and FK-DCs (2) were cultured for 9 days in the presence of FluoSpheres, and assayed by FACS on day 14. (B) anergy tests to antigen-specific CD4+ T cell lines established by KLH-pulsed DCs or KLH-pulsed FK-DCs DCs and FK-DCs pulsed with KLH were irradiated and cultured with CD4+ T cells positively selected with immunomagnetic beads from the same subjects. For generating CD4+ T cell lines, these CD4+ T cells were subsequently cultured and stimulated with irradiated autologous PBMCs as feeder cells every week for 4 weeks. For anergy tests, the proliferation of each CD4+ T cell line was evaluated by rechallenging KLH-pulsed DCs; each CD4+ T cell line (5 x 104/100 µL) was cocultured again with 50-Gy irradiated, KLH-pulsed DCs (3 x 104/100 µL) for 3 days. [3H]thymidine incorporation was measured during the final 16 h of culture. CD4+ T cell lines (T cell line 1, 2, 3) are from different donors. Each bar represents the mean ± SD of triplicates. All the P values are for comparing the [3H]thymidine uptake in primary isolated T cells in KLH-pulsed DCs (open bars) vs. KLH-pulsed FK-DCs (filled bars). *P < 0.05 compared to control by Student’s t test.

Demonstration of skewing Th2 response and reduced proliferation capacity in CD4+ T cell line established by FK-DCs
From the results concerning the uptake of latex beads (Fig. 6A) , the potential to endocytose exogenous protein should be almost the same in both groups (normal DCs and FK-DCs). We established two CD4+ T cell lines in response to KLH-pulsed DCs or KLH-pulsed FK-DCs, and assayed the ability of each to proliferate by challenging subsequently with DC-pulsed KLH in anergy tests. We found that the CD4+ T cell line generated by KLH-pulsed DCs showed effective KLH-specific proliferation, whereas CD4+ T cell line generated by KLH-pulsed FK-DCs were less effective (Fig. 6B) , suggesting that CD4+ T cells are strongly activated by normal DCs, but weakly stimulated by FK-DCs in an antigen-specific manner.

Stimulation of such cells, by either normal DCs or FK-DCs resulted in their strong reciprocal polarization. CD4+ T cell lines established by KLH-pulsed normal DCs displayed a predominant Th1 phenotype, as shown by the relative excess of IFN-{gamma} expression. CD4+ T cell lines established by KLH-treated FK-DCs, however, displayed a Th2 cytokine profile, as illustrated by the increase in IL-4 expression (Fig. 7 , Table 3 ).



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  		Figure 7. Intracellular expression of IFN-{gamma} and IL-4 by CD4+ T cell lines established by KLH-pulsed DCs or KLH-pulsed FK-DCs Each CD4+ T cell line was established in the presence of irradiated DCs (A) or FK-DCs (B), as described in the legend to Figure 6 , and intracellular cytokine expression was measured by two-color flow cytometry. Each scatter plot is representative of the result from five subjects.


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  		Table 3. Intracellular Cytokine Analysis in Antigen-Stimulated CD4+ T Cells


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DISCUSSION
 
In generating DCs by differentiation from CD34+ HPCs, we found that FK-DCs clearly belonged to the DC lineage, as shown by their morphology, as well as their phenotype. These FK-DCs displayed the same levels of expression of the costimulatory molecules, CD80 and CD86, as well as of HLA-DR as those of normal DCs (Fig. 3) . Because the previous study demonstrated that FK506 increased the number of stem cell colonies [34 ], it was shown that FK506 enhanced the number of CD1a expressing DCs in the liquid culture and our colony-forming assay (Table 2 , Fig. 3 ). Caux et al. showed that DCs can be developed from CD34+ cells by two different developmental pathways via CD1a+CD14- precursor (Langerhans cell type) or CD1a-CD14+ precursor (interstitial cell type) [35 ]. As shown in Figure 3 , FK-DCs by FK506 co-expressed CD1a as well as CD14. As previously described, CD1a+CD14+ cells may be intermediated precursor cells showing the bipotential capacity to differentiate into DCs (CD1a+CD14-) or monocytes (CD1a-CD14+) [27 ]. FK-DCs already expressed DC maturation markers, CD80, CD86, CD54, and CD40 (Fig. 3) , in addition they functionally showed a stronger allo-MLR activity than PBMCs or CD14+ monocyte/macrophages (Fig. 1A and 1B) . Taken together, they are not the macrophage lineage, but may be intermediated DCs sharing with mature DC characteristics. Early-acting growth factors (i.e., SCF and FL) together with FK506 might pool the population of CD14+CD1a+ DC by synergistic effects of GM-CSF and TNF-{alpha}.

To characterize the effects of FK506 on DC function, we compared the ability of antigen-pulsed DCs or FK-DCs to activate antigen-specific T cells. We found that FK-DCs exhibited decreased activity in allo-MLR (Figs. 1 and 4) , and that the autologous T cell proliferation induced by KLH-pulsed FK-DCs was lower than that induced by KLH-pulsed normal DCs (Fig. 6B) . After the activation by KLH-pulsed DCs, Th1-T cell line was established by the release of IL-12 [3 ]. T cell line by FK-DCs was skewed toward Th2 polarization, in line with reduced capacity of produced IL-12 than normal DCs (Fig. 5) . Lower amounts of IL-12 may be associated with a poorer antigen-presenting ability, however, only decreased IL-12 levels cannot necessarily promote IL-4 secretion by T cells. The findings may suggest the possibility of existence of the other soluble factors or cell surface-related molecules on FK-DCs.

Th1 cytokines interact to mediate the deleterious effects of acute GVHD observed after allogeneic BMT [36 ]. The balance between Th1 (IL-2 and IFN-{gamma})- and Th2 (IL-4 and IL-10)-cytokines soon after BMT may be critical for the development of acute GVHD [9 , 10 ]. It has been suggested that a Th1 to Th2 shift in the cytokine profile of donor T cells before BMT may interrupt the cascade of GVHD after allogeneic transplantation [37 ]. A shift in T helper cells from Th1 to Th2 may down-regulate the cell-mediated immune responses and inflammatory processes that occur during the course of GVHD. Administration of type 2 cytokines (e.g., direct injection of IL-4 or IL-10) after allogeneic BMT to block the development of acute GVHD, however, did not inhibit Th1 cytokine production or reduce acute GVHD [38 , 39 ]. These results suggest that the Th2 response induced by FK-DCs may potentiate a prophylactic effect on acute GVHD.

It is well known that other immunosuppressive agents, such as IL-10, corticosteroid (CS), and transforming growth factor-ß (TGF-ß) can render monocyte-derived DCs tolerogenic [40 ]. Th2 immune responses will thus be facilitated by microenvironmental factors that inhibit IL-12 production by DCs, including increased concentrations of IL-10. Both IL-10- and CS-treated DCs express low levels of IL-12 and have been shown to have a large macrophage-like morphology. In addition, these cells were found to express higher levels of CD14 or mannose receptor, but lower levels of CD1a, as well as costimulatory molecules, CD80 and CD86 than untreated DCs. IL-10-DCs and CS-DCs have endocytic activity, despite having a lower antigen-presenting capability and decreased ability to stimulate in mixed leukocyte reactions [13 14 15 16 , 18 , 19 ]. Therefore, it was demonstrated that both IL-10-DCs and CS-treated DCs derived from monocyte may induce a state of antigen-specific anergy, while retaining the characteristics of macrophages. FK-DCs described, however, possess features different from DCs treated with these immunosuppressants.

In this study, we showed that FK-DCs derived from CD34+ HPCs also highly expressed CD1a as well as CD14, and exhibit a lower production of IL-12, however, they fully expressed DC maturation markers, CD40, CD80, and CD86 as well as HLA-DR. In the functional assays, antigen-pulsed normal DCs induced Th1-type CD4+ T cells, whereas antigen-pulsed FK-DCs induced antigen-specific Th2-type CD4+ T cells, suggesting that FK506-modified DC can drive CD4+ T cells toward Th2 in vitro. Utilizing the characteristics of these subtypes of DCs may be helpful in controlling and achieving the specific Th1/Th2 response desired.


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ACKNOWLEDGEMENTS
 
This work was supported by the Sagawa Foundation for Promotion of Frontier Science. We appreciate Drs. Kyogo Ito (University of Kurume, Japan) and Michael T. Lotze (University of Pittsburgh, PA) for helpful discussion and Drs. Angus Thomson, Pawel Kalinski, Robbie B. Mailliard (University of Pittsburgh) for reviewing the manuscript. We are grateful to Dr. Kenjiro Matsuno (University of Kumamoto, Japan) for his continuous encouragement.

Received November 1, 1999; revised July 6, 2000; accepted July 7, 2000.


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