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Originally published online as doi:10.1189/jlb.1104654 on April 27, 2005

Published online before print April 27, 2005
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(Journal of Leukocyte Biology. 2005;78:383-392.)
© 2005 by Society for Leukocyte Biology

Lipopolysaccharide stimulation converts vigorously washed dendritic cells (DCs) to nonexhausted DCs expressing CD70 and evoking long-lasting type 1 T cell responses

Sanju Iwamoto*,1, Makoto Ishida*, Keiko Takahashi*, Ken Takeda{dagger} and Akira Miyazaki*

* Department of Biochemistry, School of Medicine, Showa University, Tokyo, Japan; and
{dagger} Department of Hygiene Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan

1 Correspondence: Department of Biochemistry, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan. E-mail: iwasanju{at}med.showa-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A great variety of in vitro culture protocols for human monocyte-derived dendritic cells (mo-DCs) has been used to generate DCs suitable for use in immunotherapy. It is thought that activated DCs undergo one-way differentiation into "exhausted" DCs. In the present study, we contrived an in vitro method for facilitating expression of CD70 by mature DCs. This was achieved by vigorous washing of mo-DCs before exposure to lipopolysaccharide (LPS). Unexpectedly, these mature DCs retain expression of some interleukin (IL)-12 family members after extended periods and maintain their ability to stimulate type 1 T cell responses. In contrast, DCs exposed to IL-4 before LPS stimulation or LPS-stimulated DCs not exposed to washing stress before activation failed to express CD70 and did differentiate into exhausted DCs. It is interesting that DCs expressing CD70 (CD70+ DCs) induced interferon-{gamma} production from purified, allogeneic CD8+ T cells through a direct CD27-CD70 interaction. This is evidence for a pathway resulting in generation of CD8 T effectors by B7-independent mechanisms. These data suggest that exposure of immature DCs to LPS stimulation contributes to their terminal differentiation into CD70+ DCs, which have potent ability to prolong type 1 T cell responses through alternative pathways.

Key Words: CD27 • CD88 • CTLA-4 • IFN-{gamma} • IL-4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pathogen-derived components {pathogen-associated molecular patterns (PAMPs); ref. [1 ]} have a unique role in the stimulation and differentiation of dendritic cells (DCs), which are critical to bridge innate and adaptive immunity in infection [2 ]. Through signals mediated predominantly by pattern recognition receptors including Toll-like receptors (TLRs) [3 ], DCs are stimulated to mature into cells with high-level expression of major histocompatibility complex (MHC)-peptide complexes and costimulatory molecules and with the ability to secrete immunomodulatory cytokines. These activated DCs mature without help from CD4+ T cells, although this is important for classical DC-maturation processes [3 ]. Furthermore, it is hypothesized that such DCs stimulated with PAMPs acquire a specialized ability to promote naïve T cells to differentiate into T effectors through alternative pathways [4 5 6 ]. These events contribute to the strong immune responses induced by this type of DC. Recently, it was reported that DCs stimulated by signals through TLR evoke autoimmunity [7 ] or tumor regression by overcoming tolerance [8 ].

Langenkamp et al. [9 ] developed a paradigm for lipopolysaccharide (LPS)-induced DC differentiation, which in the meantime, has become generally accepted. In their scheme, activated, immature (im)-DCs transiently evoke T helper cell type 1 (Th1) responses during the maturation process (8 h after stimulation) and convert to mature, but "exhausted" or "paralyzed," DCs at 48 h. These mature DCs prime Th2 or Th0 cells, stimulate T cell proliferation, and suppress Th1 responses [9 , 10 ]. At this stage, they would be inappropriate for DC-based immunotherapy of cancer [11 ]. Langenkamp et al. [9 ] proposed a scenario in which such exhausted DCs affect a shift of the immune response from a Th1 to a Th2 bias and prevent immunopathological tissue damage. However, such a notion is hard to reconcile with PAMP-induced autoimmune diseases [7 , 12 ] or tumor eradication [8 , 13 ], as persistent Th1 responses have important roles in both of these [14 ]. Therefore, it might be expected that an alternative fate of activated DCs is that they can differentiate into cells evoking long-lasting type 1 T cell responses.

As CD70 is a costimulatory molecule that contributes to hyperactive type 1 T cell responses [15 ] and is inducible by LPS on murine DCs [16 ], we here address the question of expression of CD70 [17 ] on human monocyte-derived (mo)-DCs stimulated with LPS. Human DCs positive for CD70 have not been reported thus far [15 , 18 ]. CD70 is a member of the tumor necrosis factor (TNF) family and is a type II transmembrane-glycoprotein expressed by activated T and B cells, which interacts with its receptor CD27 [19 ]. Its roles in the differentiation of type 1 cytolytic T lymphocytes (CTLs) [20 ] and antibody-producing cells [21 ] have been well-defined. Many other characteristics of CD70 have also been reported, such as strong induction of T effectors producing interferon-{gamma} (IFN-{gamma}) [22 ], immune suppression [15 , 22 ], the maintenance of memory T cells [23 ], and stimulation of natural killer cells [24 ]. However, the role of CD70 in priming naïve T cells by DCs has been largely unexplored, although the former expresses CD27 constitutively [19 ]. In recent reports, CD27-CD70 interactions efficiently induce strong immunity against viruses or immunotolerogenic tumors through alternative pathways [25 , 26 ]. These findings suggest a potential role of CD70+ DCs in strong immunity [27 ].

This is the first report of CD70 expressed by human activated DCs. The present study documents that LPS-induced differentiation of DCs can result in the development of CD70+ DCs, representing a novel type of cell that is not exhausted. CD70+ DCs maintain an ability to promote type 1 T cell responses long after LPS initiation, and their activation of CD8 T cells is through alternative pathways that are CD70-dependent but B7-independent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Recombinant human IFN-{alpha}, TNF-{alpha}, interleukin (IL)-1ß, granulocyte monocyte-colony stimulating factor (GM-CSF), IL-4, and IFN-{gamma} were purchased from Diaclone Research (Besançon, France). Culture grade LPS from Escherichia coli (L4516) and prostaglandin E2 (PGE2) was purchased from Sigma-Aldrich (St. Louis, MO). CD40L/Fc and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4)/Fc were purchased from R&D Systems (Minneapolis, MN). The monoclonal CD70 blocking antibody, BU69 ({alpha}-CD70), was purchased from Ancell Corp. (Bayport, MN). Keyhole limpet hemocyanin (KLH) was purchased from Wako Pure Chemical (Osaka, Japan).

Cell culture
T cells, monocytes, and mo-DCs were cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL, Auckland, New Zealand, US129924), plus 100 units/ml penicillin G and 100 µg/ml streptomycin.

Preparation of mo-DCs
Peripheral blood mononuclear cells (PBMCs) of five healthy volunteers, obtained with informed consent under the protocol approved by the ethics committee of Showa University (Tokyo, Japan), were isolated by Histopaque (Sigma-Aldrich) density gradient (1.077) centrifugation. After depletion of platelets by centrifugation with phosphate-buffered saline (PBS) three times, monocytes were isolated from PBMCs, resuspended in PBS containing 2 mM EDTA and 0.5% bovine serum albumin (BSA) by positive sorting using human anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated CD14+ cells (5x105/ml) were cultured for 7 days in the presence of GM-CSF (20 ng/ml) and IL-4 (10 ng/ml). Next, the cells were washed thoroughly five times in warm medium by centrifugation (300 g, 5 min) and subsequently cultured in the presence of LPS or the indicated cytokines, or IL-4 (10 ng/ml) was added 1 h before the addition of LPS to generate exhausted DCs. Alternatively, im-DCs were given the same volume of culture medium without washing and were stimulated with LPS. For these comparisons, stimulation was standardized to 10 µg/ml LPS for 3 days.

Preparation of T cells
Isolation of T cells from PBMCs used Pan T cell isolation kits (Miltenyi Biotec). The purity of the T cell-enriched fractions was assured by removing circulating DCs, B cells, and activated T cells using Dynabeads for human leukocyte antigen (HLA) class II and CD19 (Dynal A. S., Oslo, Norway). These purified T cell fractions were used for preparing CD4+ T cells or CD8+ T cells by positive selection with Dynabeads M-450 CD4+ T cell or CD8+ T cell reagents (Dynal A. S.). The isolated, whole T cells and the CD4+ or CD8+ subsets were confirmed to have a purity of 98% for CD3+ cells, 99% for CD4+CD3+ cells, and 99% for CD8+CD3+ cells. Additionally, naïve CD4+ T cells were purified from the CD4+ T cell fractions by positive selection of CD45RA+ cells using CD45RA magnetic microbeads (Miltenyi Biotec). Activated leukocytes positive for CD69, CD70, or CD86 were not detectable in these fractions.

Fluorescein-activated cell sorter (FACS) analysis
The cells were adjusted to a concentration of 1 x 106 cells/ml and incubated at 4°C for 30 min with appropriate antibodies. After washing twice with ice-cold PBS containing 0.3% BSA, cells were analyzed by FACS with CellQuest software (BD PharMingen, San Diego, CA). Antibodies used for flow cytometry were as follows: fluorescein isothiocyanate (FITC)-conjugated murine monoclonal antibodies (mAb) {alpha}-HLA-ABC (w6/32, Diaclone Research), {alpha}-CD4 (B-F5, Diaclone Research), {alpha}-CD8 (B-H7, Diaclone Research), {alpha}-CD27 (M-T271, BD PharMingen), {alpha}-CD70 (Ki-24, BD PharMingen), and {alpha}-CD80 (B-L2, Diaclone Research). Phycoerythrin (PE)-labeled murine mAb were {alpha}-HLA-DR (B-F1) and {alpha}-CD40 (B-B20), purchased from Diaclone Research, and {alpha}-CD86 (IT2.2), purchased from BD PharMingen.

IFN-{gamma} secretion assay
To analyze living IFN-{gamma}-producing T cells cocultured with DCs, an IFN-{gamma} secretion assay (Miltenyi Biotec) was performed. mo-DCs (3x104 cells per well), alone or mixed with purified T cells (3x105 cells per well), were cultured in RPMI 1640 containing 10% FBS for the periods indicated. The cells were then washed five times with warm culture medium, and 1 x 106 cells suspended in 90 ml culture medium were incubated with 10 ml capture antibody (supplied with the kit) at 4°C for 5 min. The cells were resuspended in 10 ml culture medium in a 15-ml plastic centrifuge tube and incubated at 37°C for 45 min with the tubes inverted every 5 min. Cells were washed twice with 0.5% BSA-PBS and were incubated with FITC-labeled anti-IFN-{gamma} antibody and other PE- or Cychrome (CyC)-labeled antibodies at 4°C for 30 min. Cells were then analyzed by FACS.

Cytokine measurement
The concentrations of IFN-{gamma}, IL-10, IL-12p70+p35, or IL-12p70+p40 in the conditioned medium of DCs and/or T cells were determined using enzyme-linked immunosorbent assay (ELISA) kits (Diaclone Research).

T cell proliferation assay
T cells (2x105 cells) were cultured alone or cocultured with DCs for 3 days. 3H-Thymidine (0.4 µCi, Amersham-Pharmacia Biotech, Buckinghamshire, UK) was added to each well, and nuclear incorporation was stopped by the addition of cold thymidine after a further 18-h culture. Nuclear isotope incorporation was determined after harvesting the cells on glass fiber sheets by liquid scintillation counting in a TracorAnalytic Mark III machine, after washing three times with PBS containing 0.3% BSA.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA from 1 x 106 cells prepared using a Nucleospin RNA II kit (BD Bioscience Clontech, Palo Alto, CA) was reverse-transcribed to synthesize single-stranded cDNA with the use of oligo-dT primer and a SMARTTM PCR cDNA synthesis kit (BD Bioscience Clontech). PCR was carried out for 33 cycles (each cycle consisting of 1 min at 60°C, 1 min at 95°C, and 1 min at 72°C) using an AmpliTaq Gold PCR kit (Roche Molecular, Pleasanton, CA). The PCR primers used in the experiments were as follows: IL-12p35: sense-GGTCTTTCTGGAGGCCAGGC, antisense-CCTCAGTTTGGCCAGAAACC; IL-12p40: sense-AAGGAAGATGGAATTTGGTCCACTG, antisense-GATGATGTCCCTGATGAAGAAGCTG; IL-23p19: sense-GAGGGAGATGAAGAGACTAC, antisense-TTTAGGGACTCAGGGTTGCT; and EBI3 primers, as designed by Hashimoto et al. [28 ]. Primers for IL-27p28 were those designed by Pflanz et al. [29 ].

Intracellular staining of cytokines in T cells and DCs
T cells (1x106 cells) were incubated with 10 ng/ml phorbol 12-myristate 13-acetate and 500 ng/ml ionomycin at 37°C for 3 h in culture medium containing 10% heat-inactivated FBS, with addition of 10 µg/ml brefeldin A (Sigma-Aldrich) 2 h before terminating the culture. Alternatively, for DCs, the cells were washed twice with ice-cold PBS and fixed in PBS containing 4% paraformaldehyde for 20 min at room temperature. Cells were washed twice with PBS containing 0.5% BSA and resuspended in permeabilization buffer (0.5% saponin, 2% FBS in PBS). After centrifugation at 1000 rpm for 2 min, cells were incubated in permeabilization buffer containing 10 µg/ml cytokine-specific antibody or control murine immunoglobulin G (IgG) for 20 min at room temperature. After washing twice with permeabilization buffer, cells resuspended in the same buffer containing 10 µg/ml FITC-labeled anti-mouse IgG were incubated for 20 min at room temperature. After centrifugation twice at 1000 rpm for 2 min in permeabilization buffer, cells resuspended in PBS containing 0.5% BSA were analyzed by FACS.

Statistical analysis
The Student’s t-test was used for analysis of in vitro data. Lack of significance was indicated by P > 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction and regulation of CD70 on mo-DCs are different from other costimulatory molecules
We analyzed conditions under which human mo-DCs express CD70 in vitro by stimulating im-DCs, which were derived from culture of CD14+ PBMCs with GM-CSF and IL-4 for 7 days. im-DCs expressed low levels of costimulatory molecules including CD40, CD80, CD86, and 4-1BBL but no CD70. We tested several DC-maturation stimuli, such as LPS, washing stress [30 ], a cocktail of cytokines (10 ng/ml IL-1ß and TNF, plus 1 µg/ml PGE2) [31 ], and combinations of these. It was found that im-DCs vigorously washed before addition of 10 µg/ml LPS expressed substantial levels of CD70 (41.1±10.6, 27.2–63.2, n=13) 3 days later (Fig. 1 ). In contrast, DCs not subjected to washing stress did not express CD70. Furthermore, DCs vigorously washed and treated with IL-4 before addition of LPS or DCs, washed and then exposed to the cytokine cocktail instead of LPS, expressed little CD70 (Fig. 1) . In contrast, all of these stimulated DCs up-regulated CD86, a marker of DC maturation (Fig. 1) . Therefore, induction of CD70 expression on DCs by LPS required washing stress and an absence of IL-4.



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  		Figure 1. Induction of CD70 on human mo-DCs. im-DCs, differentiated from CD14+ monocytes, as described in Materials and Methods, were stimulated by several treatments. Control, Cells before stimulation. Some cells were stimulated by 10 µg/ml LPS and added in the same volume of culture medium without washing. Others were washed five times with warm culture medium and stimulated by LPS in the presence or absence of 10 ng/ml IL-4 or by a cytokine cocktail (10 ng/ml IL-1ß and TNF, plus 1 µg/ml PGE2). After 3 days culture, cells were labeled with FITC- or PE-conjugated murine isotype-control IgG, FITC-labeled {alpha}-CD70, or PE-labeled CD86. Expression of CD70 on DCs was analyzed by flow cytometry. Numbers in the histograms are percent CD70+ cells. Data are representative of experiments using at least three other individuals.

 
CD70 induction by LPS increased in a time- and dose-dependent manner (Fig. 2A and 2B ). DCs stimulated by LPS up-regulated CD70 and CD86 but not in parallel. The optimal dose of LPS for CD70 expression (10–30 µg/ml) was much higher than for CD86 induction (~1 µg/ml), and the kinetics of CD70 differed from those of other costimulatory molecules, as its increase was slower (Fig. 2B) . Furthermore, CD70 expression was inhibited specifically in the presence of IL-4, and HLA-DR and other costimulatory molecules including CD40, CD80, CD83, CD86, and 4-1BBL were elevated as much as in the absence of IL-4 (Fig. 2C) . Therefore, expression of CD70 on DCs was up-regulated by LPS in a manner different from other costimulatory molecules, such as members of the B7 family, and was inhibited specifically by IL-4. Washed DCs exposed to IL-4 for 1 h before addition of LPS, which are termed IL-4/LPS-DCs, were compared with CD70+ DC in the following experiments.



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  		Figure 2. Kinetics of CD70 expression on DCs stimulated by LPS. im-DCs were washed five times with warm culture medium and then cultured with an indicated concentration of LPS. The cells were labeled with appropriate FITC- or PE-conjugated mouse mAb and were analyzed by FACS. (A) Dose-responses of CD70 expression on DCs to LPS stimuli. Negative control, Cells before stimulation. Cells were cultured in the presence of several concentrations of LPS (0.1, 1.0, 10, 30 µg/ml). After 3 days culture, cells were labeled with FITC- or PE-conjugated murine isotype-control IgG, FITC-labeled {alpha}-CD70, or PE-labeled CD86. Levels of CD70 expression on DCs were determined by FACS analysis. Numbers indicate percent CD70+ or CD86+ cells, and numbers in parentheses represent mean fluorescence intensity. (B) Time course of expression of surface antigens on LPS-stimulated DCs. im-DCs cultured with LPS for the times indicated were labeled with isotype-control IgG-FITC or -PE, {alpha}-CD70-FITC, {alpha}-CD86-PE, {alpha}-CD80-FITC, {alpha}-CD40-PE, {alpha}-HLA-ABC-FITC, or {alpha}-HLA-DR-PE. (C) Specific inhibition of LPS induction of CD70 on DCs by IL-4. im-DCs cultured with LPS in the presence or absence of 10 ng/ml IL-4 for 3 days were labeled with {alpha}-CD70-FITC, {alpha}-4-1BBL-PE, {alpha}-CD86-PE, {alpha}-CD80-FITC, {alpha}-HLA-ABC-FITC, or {alpha}-HLA-DR-PE. The numbers are percentages indicating the proportion of cells positive for these antigens. Data are representative of experiments using at least three other individuals.

 
Ability of DCs to stimulate allogeneic T cells at prolonged periods of time after LPS activation
We analyzed induction of IFN-{gamma} production in mixed cultures of allogeneic T cells and DCs different times after their stimulation with LPS to determine whether they converted to exhausted DCs. Three types of LPS-stimulated DCs were compared, i.e., vigorously washed DCs cultured in the absence (CD70+ DCs) or presence of IL-4 (IL-4/LPS-DCs) and unwashed DCs. They were stimulated with 10 µg/ml LPS for 1 day or 3 days and then cultured with allogeneic T cells for a further 3 days. As shown in Figure 3 , high levels of IFN-{gamma} were induced in all of these mixed cultures using DCs stimulated for 1 day but not 3 days. Only CD70+ DCs retained a substantial activatory capacity after 3 days. These data indicate that DCs not subjected to washing stress, as well as IL-4/LPS-DCs, lost their capacity to evoke type 1 T cell responses 3 days after stimulation, suggesting that they converted to exhausted DCs. In contrast, CD70+ DCs maintained their activatory capacity.



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  		Figure 3. CD70+ DCs maintain capacity to induce IFN-{gamma} in mixed allogeneic T cell cultures after prolonged periods of time. LPS-stimulated DCs subjected to washing stress or no washing stress before exposure to LPS and with or without IL-4 stimulation were tested. DCs (2x104 cells/well), after LPS stimulation for 1 day (open bars) or for 3 days (solid bars), were cocultured with allogeneic T cells (2x105 cells/well) for 3 days. IFN-{gamma} production in culture-conditioned medium was assayed by ELISA. The bar diagram shows the means and standard deviations of cytokine concentrations in three separate experiments. Statistical significance is indicated with CD70+ DCs and other DCs. Data are representative of experiments using at least three other individuals. N.S., Not signficant.

 
CD70+ DCs, but not IL-4/LPS-DCs, show prolonged mRNA expression and production of IL-12 family members
As significant loss of capacity of LPS-stimulated DCs to produce IL-12 later after stimulation contributes to their conversion to exhausted DCs [9 ], IL-12 family members (IL-12p35, -p40, IL-23p19, IL-27p28, and EBI3) comprising heterodimeric proteins [32 ] were assayed here. We monitored mRNA expression and production of protein in CD70+ DCs and IL-4/LPS-DCs from the beginning of culture with LPS up to 80 h later. As shown in Figure 4A , 48–80 h after stimulation, CD70+ DCs expressed mRNAs for IL-12 family members, in particular, IL-12p40 and IL-23p19, at much greater levels than IL-4/LPS-DCs. In contrast, there were no particular differences between the two, up to 24 h after LPS stimulation. Correspondingly, high levels of expression of mRNA for IL-12p35 as well as IL-27p28 were detected in IL-4/LPS-DCs and CD70+ DCs for up to 24 h but were diminished significantly at the later periods. In contrast, substantial expression of EBI3 mRNA was found in both types of DCs at all times after stimulation. These data suggest that CD70+ DCs, but not IL-4/LPS-DCs, retain substantial levels of mRNA for IL-12p40 and IL-23p19 over extended periods of time after stimulation, and both of those LPS-stimulated DCs together expressed high levels of mRNA for all IL-12 members early after stimulation. Likewise, analysis of production of IL-12p70 and IL-12p40 in cultures of 24 h LPS-stimulated DCs revealed that CD70+ DCs and IL-4/LPS-DCs produced large amounts of IL-12p40 and similar levels of IL-12p70 at this time (data not shown). According to intracellular staining (Fig. 4B) , IL-12p40 was also present in CD70+ DCs at 1 day and 4 days, and IL-4/LPS-DCs contained none 4 days after stimulation. It seems that CD70+ DCs may produce the IL-12p40 heterodimer, IL-23. Therefore, it is concluded that CD70+ DCs and IL-4/LPS-DCs do not markedly differ in their production of IL-12 family members early after stimulation, but they are quite different later. Thus, these data support the results above that CD70+ DCs are still active, and IL-4/LPS-DCs are exhausted at prolonged periods of time after activation.



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  		Figure 4. Differential expression of IL-12 family members between CD70+ DCs and IL-4/LPS-DCs. (A) Semiquantitative RT-PCR analysis of LPS-stimulated DCs (5x105 cells/ml), which were sampled during culture with 30 µg/ml LPS in the presence or absence of IL-4 before addition (0 h) and at 8, 24, 48, and 80 h after addition of LPS. Total RNAs from the samples were used for RT-PCR. Primers are described in Materials and Methods. RT-PCR was carried out in the thermal cycler (95°C, 60°C, and 72°C) for 33 cycles. G3-PDH, Glyceraldehyde 3-phosphate dehydrogenase. (B) Intracellular staining of IL-12p40 in CD70+ DCs and IL-4/LPS-DCs at 1 day and 4 days after LPS initiation. Permeabilized cells after fixation were incubated with 10 µg/ml murine IgG1 control (dotted lines) or {alpha}-IL-12p40 (solid lines) for 20 min at room temperature. After washing twice, cells were incubated with FITC-labeled donkey IgG antimurine IgG for 20 min at room temperature. After washing twice, cells resuspended in PBS containing 0.5% BSA were analyzed by flow cytometry.

 
CD70+ DCs maintain Th1 polarization
We analyzed the capacity of LPS-stimulated DCs to prime T polarization at days 1 and 3 after activation. CD70+ DCs and IL-4/LPS-DCs were exposed to KLH before stimulation with 10 µg/ml LPS. Cells cultured with LPS for the indicated times were cocultured with purified, naïve CD4+ T cells for 12 days. T cells cocultured with DCs in the presence of transforming growth factor-ß and {alpha}-IL-12 acted as a negative control. As shown in Figure 5 , CD70+ DCs and IL-4/LPS-DCs, 1 day after stimulation, engendered predominantly Th1 polarization, compared with the negative control. However, IL-4/LPS-DCs, 3 days after activation, showed a much decreased capacity, approaching that of the negative control. In contrast, CD70+ DCs still maintained substantial Th1-stimulating capacity. Taken together, these findings suggest that CD70+ DCs are not exhausted, and IL-4/LPS-DCs are exhausted.



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  		Figure 5. CD70+ DCs maintain capacity to prime Th1 cell polarization after prolonged periods of time. CD70+ DCs and IL-4/LPS-DCs were pulsed with 20 µg/ml KLH before stimulation with LPS. Cells (2x104 cells/well) were cultured for 1 day or 3 days with 10 µg/ml LPS and cocultured with naïve CD4+ T cells (2x105 cells/well) for 12 days. Intracellular cytokines produced by T cells were stained with FITC-labeled antibody to IFN-{gamma} and PE-labeled antibody to IL-4 and were analyzed by flow cytometry. Right upper and right lower panels show T effectors producing IL-4 and IFN-{gamma}, respectively. Numbers indicate percentages of cytokine-producing cells. Similar results are representative of experiments using at least three other individuals.

 
CD70 expressed on DCs plays a role in IFN-{gamma} production by CD8+ T cells
We examined the effects of CD70+ DCs on activation of CD4+ or CD8+ T cell subsets. Figure 6A shows IFN-{gamma} production in cocultures of purified, allogeneic CD4+ or CD8+ T cells and CD70+ DCs or IL-4/LPS-DCs. IFN-{gamma} production by CD4+ and CD8+ T cells stimulated by CD70+ DCs was much greater than that stimulated by IL-4/LPS-DCs. Additionally, pretreatment with {alpha}-CD70 did not block IFN-{gamma} production by CD4+ T cells but did result in reduced IFN-{gamma} by CD8+ T cells. In contrast, CD70+ DCs and IL-4/LPS-DCs stimulated proliferation of CD4+ or CD8+ T cells to a similar extent, as shown in Figure 6B . However, pretreatment with {alpha}-CD70 failed to block this. Therefore, CD70 on DCs stimulates CD8+ T cells to produce IFN-{gamma} but has no effect on CD4+ T cell activation nor on proliferation of CD4+ or CD8+ T cells.



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  		Figure 6. CD70 on DCs has a role in IFN-{gamma} production by CD8+ T cells but not in CD4+ T cell activation and T cell proliferation. CD70+ DCs and IL-4/LPS-DCs, treated or not treated with {alpha}-CD70, were cocultured with purified, allogeneic CD8+ or CD4+ T cells for 3 days. (A) CD70+ DCs affect IFN-{gamma} production by CD8+ but not CD4+ T cells. Concentrations of IFN-{gamma} in the conditioned medium were measured by ELISA. The bar diagram shows the means and standard deviations of cytokine concentrations in three separate experiments. (B) CD70 on DCs was not effective for growth stimulation of T cells. After coculturing DCs and T cells for 3 days, 1 µCi/ml 3H-thymidine was added, and cells were cultured further overnight. Cells were washed three times, and then 3H-thymidine incorporation was assayed by scintillation counting as described in Materials and Methods. The bar diagram shows the means and standard deviations of cytokine concentrations in three separate experiments. Statistical significance is for the presence versus the absence of {alpha}-CD70. Data are representative of experiments using at least three other individuals. N.S., Not significant; DPM, disintegration per minute.

 
CD70- and B7-dependent activation of CD8+ T cells by CD70+ DCs without CD4+ T cell help
It is intriguing that CD70+ DCs express B7 and CD70, which are essential for the differentiation of CD8+ T cells into CTLs. In the present experiments, CD70+ DCs were able to stimulate CD8+ T cells as a result of their expression of CD70 instead of activated CD4+ T cell, which functions in the conventional pathway [19 ]. Pretreatment of CD70+ DC with CTLA-4/Fc or {alpha}-CD70 reduced IFN-{gamma} production by approximately one-half compared with controls stimulated by alloantigens in mixed cultures of CD8+ T cells and DCs (Fig. 7A ). Furthermore, pretreatment with {alpha}-CD70 and CTLA-4/Fc together reduced IFN-{gamma} production further by ~80% relative to controls. These results suggest that CD70 and B7 (CD80 and CD86) contributed to stimulating IFN-{gamma} production following recognition of alloantigens by CD8+ T cells on the CD70+ DCs.



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  		Figure 7. CD70- and B7-independent activation of CD8 T cells by CD70+ DCs, obtained by the culture of im-DCs with 10 µg/ml LPS after washing stress. DCs (2x104 cells/well) were pretreated with 10 µg/ml {alpha}-CD70 or/and CTLA-4/Fc at 6–12°C for 1 h and cultured with purified allogeneic CD8+ T cells (CD8+>99%) for 2 days. (A) IFN-{gamma} production in culture-conditioned medium of CD8 T cells combined with DCs was assayed by ELISA. The bar diagram shows the means and standard deviations of cytokine concentrations in three separate experiments. Statistical significance is for the presence versus the absence of {alpha}-CD70. (B) Analysis of IFN-{gamma}-secreting CD8+ T cells activated by CD70+ DCs and alternation of CD8+ T cell differentiation stage on activated CD8 T cells by CD70 on DCs. Black dots in the figure indicate IFN-{gamma}-producing cells, and numbers in squares are percentages of IFN-{gamma}-producing cells in each CD8+ T cell differentiation stage (dot blots). IFN-{gamma} secretion was determined as described in Materials and Methods. Cells labeled with {alpha}-IFN-{gamma}-FITC, {alpha}-CD27-PE, and {alpha}-CD28-CyC were assayed by flow cytometry. Numbers below squares are percentages of CD27+CD28+ cells, CD27–CD28+ cells, CD27+CD28– cells, and CD27–CD28– cells. Data are representative of experiments using at least three other individuals.

 
We analyzed how CD70+ DCs stimulate CD8+ T cells via CD70 and B7 early during DC-T cell interactions. T cells stimulated by their ligand transiently down-regulate these surface antigens, as CD27 is dissociated from the surface membrane [33 ], and CD28 is down-regulated after ligation [34 ]. We measured these parameters at day 2, as recovery of expression had not yet occurred at this time, but IFN-{gamma} production was already detectable. As a putative model for T cell differentiation [35 ], CD27+CD28+ cells as precursors or early-differentiated cells progress to CD27–CD28– T cells through several intermediate stages. In this system, increases of CD8+ T cells at each differentiation stage, namely CD27+CD28+, CD27+CD28–, CD27–CD28+, and CD27–CD28–, were monitored in cocultures with CD70+ DCs or IL-4/LPS-DCs (CD70-negative). The effects of CD70 expressed by DCs on the differentiation of CD8+ T cells to each stage were examined by pretreating with {alpha}-CD70 antibody. Figure 7B shows that CD70+ DCs exerted marked CD70-dependent effects on CD8 T cell differentiation to effectors producing IFN-{gamma}. CD70+ DCs caused CD27 down-regulation on CD8 T cells, resulting in a decrease in CD27+CD28+ cells and increases in CD27–CD28+ and CD28–CD27– cells, in contrast to IL-4/LPS-DCs, which did not cause such changes. There was an increase of IFN-{gamma}-producing cells in these populations of CD27–CD28+ and CD28–CD27– cells. Additionally, these changes were inhibited by pretreatment with {alpha}-CD70. In contrast, IL-4/LPS-DCs had only slight effects on CD27 down-regulation and increases in IFN-{gamma}-producing cells, and inhibition by {alpha}-CD70 was minimal. Furthermore, {alpha}-CD70 treatment did not interfere with the down-regulation of CD28 by B7 and neither did CTLA-4/Fc treatment affect CD27 down-regulation by CD70 (data not shown). Thus, it is concluded that CD70+ DCs have a crucial role in alternative pathways of differentiation of CD8 T cell effectors producing IFN-{gamma} in a B7-CD28-independent manner.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we present the first evidence that human im-DCs, washed vigorously before exposure to LPS, do express CD70 and differentiate into DCs, which maintain the capacity to generate T effectors, producing IFN-{gamma}, even 3–4 days after their activation. These findings imply that the fate of CD70+ DCs is quite different from that of activated, CD70-negative DCs, which convert to exhausted DCs, and that CD70 expression itself is a hallmark, functional phenotype of CD70+ DCs. Furthermore, CD70 expression has an important role in alternative generation of CD8 T effectors producing IFN-{gamma}.

It is interesting that the induction of CD70 was amplified specifically by LPS but regulated by IL-4, unlike other costimulatory molecules. These results therefore imply a specific role of CD70 on DCs. As environmental instruction of im-DCs influences their differentiation into DCs having Th1-inducing capacity [36 ], the vigorous washing of these DCs before addition of LPS may contribute to the differentiation that results in induction of type 1 T cell responses long after initial DC activation. Presumably, the manipulations during thorough washing give stress to im-DCs and affect their later differentiation [12 ]. Alternatively, removing some inhibitors of DC maturation, such as IL-10 [37 ], may also have contributed. In the fact, Langenkamp et al. [9 ] reported that IL-10, contained in DC culture medium, contributed to their exhaustion when DCs were unwashed. It is interesting that IL-4 and IL-10 also prevent CD70 expression [38 , 39 ]. As IL-4 stimulation was also found to contribute to exhaustion of DCs in the present study, these results imply that Th2 cytokines cause CD70 suppression and DC exhaustion and thereby inhibit type 1 T cell responses. Thus, effects of Th2 cytokines on im-DC would have a crucial role in determining whether they become exhausted and consequently evoke Th0 or Th2 rather than responses.

CD70+ DCs facilitated a unique pathway of differentiation of CD8+ T effectors producing IFN-{gamma}, i.e., into CD27–CD28+CD8+ effectors in a CD70-dependent manner. It is intriguing that the effectors were not stimulated by B7, and part of the inducing capacity of CD70+ DCs was resistant to blockade of CTLA-4 (Fig. 7A) , which otherwise plays a major role in the induction of peripheral tolerance and the direct regulation of CTL generation [40 ]. This might be a possible pathway to generate type 1 CD8 T effectors bypassing CTLA-4 regulation. Therefore, CD70-dependent IFN-{gamma} production by CD8+ T cells, following activation by CD70+ DCs, might augment type 1 T cell responses and thereby induce the hyperactive responses seen in CD70-transgenic mice [15 ]. More recently, Bullock and Yagita [41 ] reported that CD70, expressed on DCs, contributes to primary CD8 T cell expansion and fully functional memory CD8 T cells without the help of CD4 T cells in MHC class II-deficient mice. In conclusion, CD70+ DCs have a potential ability to initiate alternative immune responses to generate effector/memory CD8 T cells.

In contrast to CD8 T cell activation, CD4 T cells were not induced to differentiate into IFN-{gamma}-producing effectors by CD70 stimulation, although CD70+ DCs had much stronger effects on CD4 T cells than IL-4/LPS-DCs. Although direct stimulation via CD27-CD70 interactions did influence CD4 T cell activation, CD40-CD40L interactions cause down-regulation of CD27 and interfere with CD27-CD70 [39 , 42 ]. CD40L is expressed on CD4 T cells but not CD8 T cells. Alternatively, we reported that human Langerhans cell-like cells promote CD8 T cells but not CD4 T cells to produce IFN-{gamma} in a CD70-dependent manner [43 ]. The mechanism is quite different from IFN-{gamma} production by CD4 T cells, as IL-12 is unnecessary. Therefore, we speculate that such mechanisms may contribute to differences between these T cell subsets and that molecules other than CD70, including IL-12 family members, may cause CD4 T cell activation by CD70+ DCs.

Our data suggest that CD70+ DCs expressed IL-23 rather than IL-12p70. It is intriguing that IL-23 not only has similar effects to IL-12p70 on induction of Th1 responses but also certain differences. These include the maintenance of Th1-inducing capacity of DCs [44 ], memory T cell activation [45 ], induction of autoimmune inflammation [46 , 47 ], and generation of CTLs specific for tumor antigens [48 ]. Such activities might be features of CD70+ DCs, distinguishable from conventional-activated DCs. However, the precise details of IL-23 production and function in CD70+ DCs remain to be clarified.

In conclusion, our findings reveal a novel type of mature DC, a CD70+ DC, which is generated by vigorously washing im-DCs before stimulation with LPS. These cells retain the capacity to evoke type 1 T cell responses long after LPS initiation and contribute to an alternative pathway of T cell differentiation into CD8+ effectors, producing IFN-{gamma} as a result of direct CD27-CD70 interactions, independently of CD28-B7. Many features of CD70+ DCs remain to be elucidated. It is interesting that recent data from murine models showed that CD70, including CD70+ DCs, has important roles in establishing strong immunity through alternative pathways. These results are consistent with our data in humans presented here. It is anticipated that the alternative, CD70-dependent pathway will prove useful for DC-based immunotherapy and will provide a new avenue for dissecting mechanisms of autoimmunity or tumor regression involving PAMP-activated DCs [49 , 50 ].


   ACKNOWLEDGEMENTS
 
The authors thank Professor Kouji Matsushima for his critical comments on the manuscript and helpful discussion and Dr. Shinichi Hashimoto for providing EBI3 primers.

Received November 12, 2004; accepted April 7, 2005.


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 ABSTRACT
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 MATERIALS AND METHODS
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 DISCUSSION
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