Skip Navigation


International Immunology Advance Access originally published online on June 13, 2006
International Immunology 2006 18(8):1253-1263; doi:10.1093/intimm/dxl058
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow Supplementary Data
Right arrow All Versions of this Article:
18/8/1253    most recent
dxl058v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ito, H.
Right arrow Articles by Miyajima, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, H.
Right arrow Articles by Miyajima, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

IL-18 produced by thymic epithelial cells induces development of dendritic cells with CD11b in the fetal thymus

Hiroaki Ito1,2, Eiji Esashi1,2, Taishin Akiyama3, Jun-ichiro Inoue3 and Atsushi Miyajima1,2

1 Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Kawaguchi, Japan
3 Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Correspondence to: A. Miyajima; E-mail: miyajima{at}iam.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Thymic dendritic cells (DCs) are suggested to be involved in T cell selection; however, their exact origin and function remain to be established. Although DCs in the adult thymus are mostly CD8{alpha}+CD11b–, we found that CD8{alpha}–CD11b+ DCs were abundantly present in the fetal thymus and they possessed antigen-presenting activity. Interestingly, these CD11b+ DCs were significantly decreased in mice deficient for TNFR-associated factor 6 (TRAF6), a key signaling molecule downstream of IL-1 and tumor necrosis factor-{alpha} that have been known to induce DCs from intra-thymic precursor cells. CD11b+ DCs were induced from CD4–CD8– thymocytes by fetal thymic epithelial cells (TECs). Analysis of cytokine expression in TECs revealed that none of the cytokines previously shown to induce DCs were expressed. Instead, we found strong expression of IL-18 that transmits signals through TRAF6. IL-18 induced CD11b+ DCs from CD4–CD8– thymocytes in vitro, which exhibited strong antigen-presenting activity and formed conjugates with CD4+CD8+ T cells efficiently. Taken together, these results strongly suggest that CD11b+ DCs are differentiated from CD4–CD8– thymocytes by IL-18 produced from TECs and that they are involved in T cell selection in the fetal thymus.

Keywords: CD11b, dendritic cells, fetal thymus, IL-18, thymic epithelial cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) and play crucial roles for the induction of T cell immunity as well as control of B cells and NK cells (1). DCs act as a sentinel in peripheral tissues, continuously sampling antigens, and initiate immune responses by presenting antigens to T cells upon encounters with microbial products or tissue damages (2, 3). In contrast, DCs in the thymus are involved in central tolerance of T cells, which is achieved by inducing apoptosis of self-reactive T cells (4). A variety of DC subsets have been described based on their cell surface phenotype and morphology and are widely distributed in various tissues including lymphoid and non-lymphoid tissues (5). However, the developmental origin of DC subtypes has been a controversial issue (6). Differences in the cytokine and transcriptional factor requirements suggest different developmental pathways for DCs (7–12).

In the adult mouse thymus, most DCs are CD8{alpha}+CD11b– (13–15), but DCs in the fetal thymus are negative for CD8{alpha} (16). A unique feature of thymic DCs is that they can be derived from intra-thymic T/DC precursors and stay in the thymus. It has been shown that T cell precursors have a potential to differentiate into DC in liquid culture and fetal thymus organ culture (FTOC) in the presence of cytokines, or by intra-thymic/intravenous transfer (17–20). However, it has also been shown that the development of thymic DC is independent of Notch 1, which is required for early T cell development (21), and that the development of thymocytes and thymic DCs is dissociated in c-kit/{gamma}c-double deficient and RAG2-deficient mice (22). Also, in the adult mouse thymus, DCs or their precursors are supplied from bloodstream under the steady state (23). Therefore, the origin of thymic DCs and factors involved in the DC development still remain unclear. While thymic DCs are involved in the establishment of central tolerance including negative selection (4, 24), a major issue is whether thymic DCs possess distinct immunologic functions or whether their function is determined largely by environmental cues (25). To address this issue, it is necessary to better understand the nature of thymic DCs.

In this report, we describe a novel subset of thymic DCs, CD8{alpha}–CD11b+ DCs, in the fetal thymus. These cells exhibit strong APC activity and are reduced in TNFR-associated factor 6 (TRAF6)-deficient fetal thymus, suggesting a cytokine that activates TRAF6 is required for thymic DC development. We show that thymic epithelial cells (TECs) express IL-18 and that they induce the differentiation of CD11b+ DC from CD4–CD8– thymocytes. Finally, we show that the IL-18-induced DCs exhibit strong APC activity and preferentially bind CD4+CD8+ T (DPT) cells. These results suggest that IL-18 derived from TECs induces the development of a novel class of thymic DCs in the fetal thymus.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Mice
C57BL/6 and BALB/c mice were purchased from SLC (Shizuoka, Japan). The generation of TRAF6-deficient mice was described previously (26). The time at midday was taken to be embryonic day (E) 0.5 for the plugged mice. All mice were housed in specific-pathogen-free-barrier animal facilities. All experiments were performed according to our institutional guidelines.

Antibodies and reagents
The following antibodies were used: rat IgG isotype control (R35-95), rat IgM isotype control (R4-22), hamster IgG isotype control (G235-2356), anti-Fc{gamma}R (2.4G2), anti-CD11c (HL3), anti-I-Ab (AF6-120.1), anti-CD40 (3/23), anti-CD86 (GL-1), anti-CD8{alpha} (53-6.7), anti-CD11b (M1/70), anti-CD4 (GK1.5), anti-CD3{varepsilon} (145-2C11), anti-CD25 (7D4), anti-CD44 (IM7) and anti-Thy1.2 antibody (30-H12). All these antibodies were purchased from BD PharMingen (San Diego, CA, USA). Anti-IL-18R {alpha} chain (IL-18R{alpha}) antibody was purchased from R&D systems (Minneapolis, USA). Streptavidin–allophycocyanin was purchased from Molecular Probes (Eugene, OR, USA), recombinant murine (rm) stem cell factor (SCF) (100 ng ml–1) and rmIL-7 (10 ng ml–1) were purchased from PEPROTECH (London, UK), rmIL-1ß (5 ng ml–1) and rm tumor necrosis factor (TNF)-{alpha} (5 ng ml–1) were purchased from WAKO (Osaka, Japan), rmIL-18 (12.5 ng ml–1) was purchased from MBL (Nagoya, Japan) and oncostatin M (OSM) (10 ng ml–1) was purchased from R&D systems.

Flow cytometry and cell sorting
Thymi were disaggregated and filtrated through a 40-µm mesh. Cells suspended in PBS containing 2% fetal bovine serum (FBS) and 2 mM EDTA were pre-treated with blocking antibody against Fc{gamma}R to eliminate non-specific staining. They were then incubated with various antibodies or appropriate isotype-matched control antibodies. After 30 min incubation, cells were washed with PBS and incubated with 7-amino-actinomycin D (7-AAD) (BD PharMingen). After a wash with PBS, cells were suspended in PBS and analyzed by FACSCalibur (BD Bioscience). Dead cells were excluded by 7-AAD staining. Cell sorting was performed with FACSVantage SE (BD Bioscience).

Preparation of primary TECs
Primary TECs were prepared as described (27). In brief, E16.5 thymi were finely minced and slowly stirred in PBS for 30 min at 4°C to release the bulk of free thymocytes and then digested in a mixture of dispase–collagenase three times for 30 min at 37°C. TECs were precipitated by centrifuging at 800 revolutions per minute and then inoculated into gelatin-coated 12-well plate (7.5 embryos per well) and cultured for 2 weeks in the standard culture medium.

In vitro culture of fetal thymocytes
The culture medium we used was RPMI 1640 (GibcoBRL) supplemented with 10% FBS, 50 µM 2-mercaptoethanol and gentamicin sulfate (the standard culture medium). Thymocytes or sorted double-negative (DN) 1 and DN2 cells from E14.5 fetal thymi were cultured for 6 days in the presence of IL-7 and SCF with or without condition medium (CM) of primary TECs or OSM-responsive TEC line (ORTEC) (28), IL-18 or a combination of IL-1ß, IL-3 and TNF-{alpha}.

Mixed lymphocytes reaction
Thymic DCs, fetal thymocyte-derived DCs, splenic DCs and LPS-stimulated DCs were used as APCs. Fetal thymocyte-derived DCs were prepared from E14.5 C57BL/6 thymocytes by culture with ORTEC-CM or IL-18 and the CD11c+I-Ab+ population was sorted by FACSVantage SE. Splenic DCs and LPS-stimulated DCs, which were prepared from mice injected with LPS (100 µg per body), were isolated from C57BL/6 spleen by positive selection with the AutoMACS using mAb to CD11c. These APCs were co-cultured with 2 x 105 naive CD4+ T cells isolated from BALB/c spleen by positive selection with the AutoMACS using mAb to CD4. Purified T cells were cultured with {gamma} ray-irradiated (30 Gy) APCs for 5 days. The culture medium consisted of RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acid, 50 µM 2-mercaptoethanol, 10 mM HEPES and gentamicin sulfate (the DC culture medium). T cell proliferation was measured using the WST-1 system (Roche, Switzerland) and the OD450/650 was measured after 4 h using a microplate reader.

Reverse transcription–PCR
Total RNA prepared by using TRIZOL (Invitrogen) was reverse transcribed with the first-strand cDNA synthesis kit (Amersham Pharmacia) and PCR was performed using the AmpliTaq DNA polymerase kit (Applied Biosystems). Primer sets used were as described previously (28–30). After incubation for 2 min at 94°C, PCR amplification was performed using the GeneAmp PCR system (Perkin Elmer) under the following conditions: denaturation at 94°C for 30 s, annealing at the temperature as described in previous reports (28–30) for 30 s and elongation at 72°C for 1 min. The amplified PCR products were electrophoresed on a 2% agarose gel, and visualized with ethidium bromide. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control.

ELISA
ORTEC was cultured under the standard conditions for DC induction and culture supernatants were recovered. Primary TEC culture supernatant was recovered as described. The supernatants were used for ELISA with the murine IL-18 ELISA kit (MBL) according to the manufacturer's protocol.

RNase protection assay
The RNA probe was made by using mCK-2b multi-probe template sets (BD Bioscience) and labeled with [{alpha}-32P]uridine 5'-triphosphate by in vitro RNA transcription according to the manufacturer's directions. Total RNA was recovered by using TRIZOL (Invitrogen) from E14.5, E16.5, E18.5, P1 and adult thymi. Each 20 µg of RNA was hybridized with 32P-labeled RNA probe (7.9 x 105 counts per minute per sample). RNase treatment was then performed using RNase A and RNase T1. After RNase treatment, samples were electrophoresed. The dried gel was then placed on an Imaging Plate (BAS-MS2040; FUJIFILM, Japan) in a cassette and developed for 12 h at room temperature. The developed Imaging Plate was analyzed with a BAS-1500 bio-imaging analyzer (FUJIFILM).

T/DC conjugation assay
Thymic DCs (CD11c+CD11b+ or CD11c+CD11b– cells) and CD4+CD8{alpha}+ T cells (DPT) were isolated from E16.5 thymi using a cell sorter. IL-18-induced DCs or IL-1ß-induced DCs were produced from E14.5 thymocytes and the CD11c+ population was recovered using a cell sorter. In all cell sorting, dead cells were excluded by 7-AAD staining. These DCs were inoculated into a 96-well round-bottom plate and cultured for 15 h with DPT cells at a DC: T cell ratio of 1:2 (10 000 cells:20 000 cells per well) in the presence of SCF and IL-7 in the DC culture medium. After 15 h cultivation, cells were gently collected with the cell dissociation buffer and washed with PBS containing 2% FBS. Recovered cells were incubated with Fc{gamma}R antibody to eliminate non-specific staining and then stained with CD3 antibody and CD11c antibody. Cells were analyzed by FACSCalibur to detect CD3+CD11c– T cells, CD3–CD11c+ DCs and CD3+CD11c+ T/DC conjugates. Dead cells were excluded by 7-AAD staining. The conjugation of T cells and DCs was calculated from the number of CD3+CD11c+ conjugates among viable CD11c+ cells.

Statistics
Data were analyzed by Student's t-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
CD8{alpha}–CD11b+ DCs in the fetal thymus
T cell development takes place actively in the fetal thymus and requires the specific thymic microenvironment. Thymic DCs are an important component of the microenvironment and it is well established that DCs in the adult thymus express CD8{alpha} but not CD11b. While CD8{alpha} expression was a basis for the proposal of ‘lymphoid DC’, Dakic et al. (16) recently showed that DCs in the fetal thymus are negative for CD8{alpha}. We found that CD8{alpha}– fetal thymic DCs are further divided into CD11b– and CD11b+ populations (Fig. 1A). While almost 50% of CD11c+ cells in E16.5 thymus were CD11b+, the fraction of CD11b+ cells decreased after birth and became a minor population in the adult thymus (~5%) (Fig. 1B). MHC class II (I-Ab)-positive DCs (CD11c+I-Ab+) in E16.5 thymus mostly expressed CD11b (Fig. 2A). Moreover, expression of CD86, a co-stimulatory molecule, was stronger in CD11b+ DCs than CD11b– DCs in the fetal, but not in adult thymus (Fig. 2B). Consistently, CD11b+ DCs in the fetal thymus exhibited APC activity by the allogenic mixed lymphocytes reaction (MLR), similar to that of CD11b– DCs in the adult thymus (Fig. 2C). Taken together, these results suggest that CD11b+ DCs are functionally maturated as APCs and are involved in T cell selection in the fetal thymus.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1 Phenotype of fetal and adult thymic DCs. (A) FACS analysis of thymic DC. In E16.5 thymus, CD8{alpha}+CD11c+ DC, a typical adult thymic DC, was barely detected and CD11c+ cells were divided into CD11b+ and CD11b– populations. (B) Profiles of DC populations in the thymus. The CD11b+ cells were a major population in the CD11c+ cells in the fetal thymus and declined in post-natal thymus.

 

Figure 2
View larger version (39K):
[in this window]
[in a new window]
  		Fig. 2 Characterization of CD11b+ DCs in the fetal thymus. (A) FACS analysis of E16.5 thymus. E16.5 thymocytes were stained with antibodies against CD11c, I-Ab and anti-CD11b. Left panel, expression of CD11b in CD11c+I-Ab– cells or CD11c+I-Ab+ cells. Right panel, expression of I-Ab in CD11c+CD11b– cells or CD11c+CD11b+ cells. Most of CD11c+I-Ab+ cell, but not CD11c+I-Ab– cells, were CD11b positive in E16.5 thymus, suggesting higher APC activity in CD11c+CD11b+ cells than CD11c+CD11b– cells. (B) Expression of CD86. Fetal and adult thymocytes were stained with antibodies against CD11c, CD11b and CD86. Panels show CD86 expression in either CD11c+CD11b– cells or CD11c+CD11b+. CD86 was expressed much strongly in CD11b+ DCs than CD11b– DCs in E16.5 thymus. CD86 expression in E16.5 CD11b+ cells was also stronger than that in adult CD11b+ cells. (C) APC activity of thymic DCs by the allogenic MLR assays. Splenic T cells were mixed with CD11b+ or CD11b– DCs from E16.5 and adult thymus and T cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. T cell proliferation without stimulation was used to normalize the relative APC activity of CD11b+ DCs in E16.5 thymus, exhibited APC activity as strong as adult CD11b– DCs, typical adult thymic DCs. Error bars indicate standard deviation. *P < 0.02.

 
TRAF6 signaling is required for DC development in the fetal thymus
IL-1 and TNF-{alpha} have been known to induce DCs from thymic precursor cells in vitro (19, 20) and these cytokines activate the nuclear factor-{kappa}B signal cascade through the TRAF6 molecule (31), suggesting that TRAF6 is important for DC development from thymocytes. In fact, Kobayashi et al. (32) recently reported that TRAF6 is essential for the development and maturation of DCs in the adult spleen. We therefore considered the possibility that a cytokine activating the TRAF6 signaling pathway might be also responsible for the development of DCs in the fetal thymus. Although there was no significant difference in the size of thymi between wild-type (WT) and TRAF6-deficient mice, we found that thymic CD11b+ DCs were dramatically reduced in TRAF6-deficient mice compared with WT mice (Fig. 3A and B), whereas no such difference was found in the thymic CD11b– DCs between WT and TRAF6-deficient fetal thymus (Fig. 3C). Therefore, a factor activating the TRAF6 signaling pathway is required for the development of CD11b+ DCs in the fetal thymus.


Figure 3
View larger version (26K):
[in this window]
[in a new window]
  		Fig. 3 DC populations in TRAF6-deficient thymus. FACS analysis of TRAF6-deficient E16.5 thymus. Thymocytes from WT and TRAF6 KO mice at E16.5 were stained with antibodies against CD11c and CD11b. CD11c+ cell numbers were significantly reduced in TRAF6 KO mice. Thymic CD11c+ cells were divided into two populations using anti-CD11b antibody. DC numbers were counted by flow cytometric analysis. Thymic CD11b+ DC numbers were significantly reduced in TRAF6 KO mice, while no such change was observed for thymic CD11b– DCs. Results are representative of two independent experiments. Error bars indicate standard deviation. *P < 0.02, **P > 0.1.

 
Development of CD11b+ DCs is induced from fetal thymocytes by TECs
As thymic DCs are believed to derive from intra-thymic precursors, we considered the possibility that the thymic environment plays a key role for the development of CD11b+ DCs. As TECs are essential for thymic development, we tested whether TECs are involved in DC development. We prepared TECs from E16.5 fetal thymi and cultured for 2 weeks to establish ‘primary TEC’ (Fig. 4A) and the culture medium was added to CD4–CD8– thymocytes from E14.5 thymus in the presence of IL-7 and SCF. After 6 days of incubation, a population of CD11c+CD11b+ cells developed in this culture, which expressed I-Ab and CD86 (Fig. 4B–D). This result suggested that primary TECs secreted a factor that induces the development of CD11b+ DCs from DN thymocytes. As we previously established a TEC line, ORTEC, from fetal thymus (28), we examined the possibility that CD11b+ DC development is also supported by ORTEC. The addition of ORTEC-CM to CD4–CD8– thymocytes also induced the development of CD11c+CD11b+ cells with I-Ab, CD86 and CD40 (Fig. 5A). The APC activity of CD11c+CD11b+ cells generated by ORTEC-CM was much higher than that of freshly isolated splenic DCs and was equivalent to that of LPS-stimulated splenic DCs (Fig. 5B), indicating that the CD11c+CD11b+ cells generated in vitro by ORTEC-CM are functional DCs. As CD11b+ DCs were generated in the culture with ORTEC-CM, it was likely that a cytokine utilizing the TRAF6 signaling pathway was also involved in the development of DCs in vitro.


Figure 4
View larger version (57K):
[in this window]
[in a new window]
  		Fig. 4 Primary TECs induce DC development from CD4–CD8– thymocytes. (A) Morphology of primary TECs. Freshly isolated TECs from E16.5 thymus were cultured as described in Methods. The picture was taken by Nikon TMS-F microscope (Nikon, Chiyoda, Japan). Original magnification, x20. (B–D) Induction of the CD11c+CD11b+ population from CD4–CD8– thymocytes by TEC. E14.5 thymocytes cultured with TEC-conditioned medium for 6 days in the presence of IL-7 and SCF. Cultured cells were stained with antibodies against CD11c, CD11b, I-Ab, CD86 and CD40.

 

Figure 5
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 5 ORTEC induce DC development from CD4–CD8– thymocytes. (A) Induction of the CD11c+CD11b+ population from CD4–CD8– thymocytes by TEC. E14.5 thymocytes cultured with ORTEC-CM for 6 days in the presence of IL-7 and SCF. Cultured cells were stained with antibodies against CD11c, CD11b, I-Ab, CD86 and CD40. (B) Antigen-presenting activity detected by allogenic MLR assay. CD11c+I-Ab+ cells induced in vitro by ORTEC-CM-stimulated allogenic T cells. APC activity of ORTEC-CM-induced DCs (closed triangles) was at the same level as LPS-stimulated splenic DCs (closed squares). PBS-treated splenic DCs (open circles) were used as a negative control. Results are representative of two independent experiments. All experiments were performed in triplicate and error bars indicate standard deviation.

 
Differentiation of CD4–CD8– thymocytes proceeds through distinct stages characterized by the expression of CD44 and CD25, i.e. CD44+CD25– (DN1), CD44+CD25+ (DN2), CD44–CD25+ (DN3) and CD44–CD25– (DN4). It is known that T cell precursors including DN1 and DN2 cells have a potential to differentiate into DCs in the liquid culture, FTOC or intra-thymic/intravenous transfer (17–20). To examine whether ORTEC-CM induces DC development from DN1 and DN2 cells, DN1 and DN2 cells isolated from E14.5 thymus were cultured with IL-7 and SCF in the presence or absence of ORTEC-CM. While both populations proliferated in this culture, DN1 cells proliferated better in the presence of ORTEC-CM than without ORTEC-CM (Fig. 6A). FACS analysis revealed that both DN1 and DN2 cells produced CD11c+CD11b+ cells only in the presence of ORTEC-CM (Fig. 6B and C). As DN1 cells produced more DCs than DN2 cells, the potential to differentiate to DCs may be gradually reduced during the transition from the DN1 to DN2 stage.


Figure 6
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6 The origin of DCs induced in vitro by ORTEC-CM. (A) ORTEC induces DC development from CD4–CD8– thymocytes. CD44+CD25– (DN1) or CD44+CD25+ (DN2) cells isolated from E14.5 thymi were cultured with or without ORTEC-CM for 6 days in the presence of IL-7 and SCF. Cultured cells were counted. The fold expansion is relative to the number of input cells in this culture. (B) DN1 and DN2 cells were cultured with or without ORTEC-CM. Cultured cells were stained with antibodies against CD11c and CD11b, and analyzed by flow cytometry. Note that ORTEC-CM, but not a combination of IL-7 and SCF, specifically induced the differentiation of DCs from DN cells. (C) DC numbers were evaluated by flow cytometric analysis. The DC population (CD11c+CD11b+) was generated from both DN1 and DN2 cells by ORTEC-CM. Results are representative of three independent experiments. All experiments were performed in triplicate and error bars indicate standard deviation.

 
Identification of a factor required for DC development
To identify a factor produced by ORTEC which induces the differentiation of CD11b+ DCs from thymocytes, we analyzed the expression of cytokines known to stimulate DC development or to maintain survival of thymocytes by using microarray (data not shown) and reverse transcription (RT)–PCR. Among various cytokines we examined, only IL-18, which is known to activate the TRAF6 signaling pathway, was clearly expressed in ORTEC (Fig. 7A). None of the other cytokines, IL-1ß, IL-3, IL-4, IL-12, TNF-{alpha}, granulocyte macrophage colony-stimulating factor and Flt-3L, which are also known to stimulate DC development (19, 33–35), was detected in ORTEC (Fig. 7A). As IL-18 is a member of the IL-1 cytokine family and has been known to activate DCs in the periphery (36), we examined IL-18 expression in more detail. IL-18 was expressed in primary cultured TECs as well as freshly isolated TECs from E16.5 thymus (Fig. 7B). Furthermore, significant production of IL-18 protein was detected in the culture medium of both primary TECs and ORTEC by ELISA (Fig. 7C). To assess the expression of IL-18 in the thymus, we performed the RNase protection assay. IL-18 was detected in the thymus from fetus to adult (Fig. 7D). Consistently with the results for ORTEC, IL-1ß expression was not detected in the thymus. Inflammatory cytokines such as IL-6, IL-12, IFN-{gamma} and IL-1{alpha} were undetectable or barely detectable in the thymus. Interestingly, IL-1R antagonist, which is a specific inhibitor of IL-1 (37, 38), was constitutively expressed at a high level in the thymus (Fig. 7D). These results may support the notion that inflammatory responses do not occur in the normal thymus, allowing T cells to develop without interference. In contrast, the IL-18 level was significantly high during thymic development, suggesting that IL-18 plays an important role in thymic microenvironment and is involved in CD11b+ DC development from thymocytes.


Figure 7
View larger version (39K):
[in this window]
[in a new window]
  		Fig. 7 Expression of cytokines in various TECs and thymus. (A) Expression of cytokines, which were known to be involved in the DC development, was evaluated in ORTEC by semi-quantitative RT–PCR. (B) RT–PCR analysis for the expression of IL-18 in freshly isolated TECs from E16.5 thymus by cell sorting of the CD45 negative fraction (indicated as ‘F’) and primary TECs (indicated as ‘P’). (C) Production of IL-18 protein by primary TECs and ORTEC was evaluated by ELISA. IL-18 was secreted from both primary TECs and ORTEC. Experiments were performed in triplicate and error bars indicate standard deviation (N.D., the background level of IL-18 production). (D) Expression of various cytokines in the thymus during ontogeny. The expression of cytokines as indicated was evaluated using the RNase protection assay. Expression of IL-18, IL-1R antagonist and IL-1{alpha} was detected in the fetal thymus, while other inflammatory cytokines were undetectable or barely detectable.

 
We examined the expression of IL-18R subunits in DN1 and DN2 cells isolated from E14.5 thymus (Fig. 8A). In order to avoid contamination of resident DCs and myeloid cells, CD11c+ cells were depleted from the DN1 and DN2 cells. RT–PCR analysis revealed that both IL-18R{alpha} and IL-18Rß were expressed in DN1 and DN2 cells but IL-1R (IL-1R1) was not detected in DN1 or DN2 cells (Fig. 8B). Furthermore, we demonstrated IL-18R{alpha} protein expression on the cell surface of DN1 and DN2 cells (Fig. 8C). These results strongly suggest that IL-18 derived from TECs directly acts on CD4–CD8– thymocytes and induces differentiation to CD11b+ DCs.


Figure 8
View larger version (54K):
[in this window]
[in a new window]
  		Fig. 8 IL-18R expression on DN1 and DN2 cells. (A) Fractionation of E14.5 thymus into DN1 and DN2 cells by using CD44 and CD25 antibodies. (B) RT–PCR analysis was performed to detect the expression of IL-18Rs (IL-18R{alpha} and IL-18Rß) in DN1 and DN2 cells. IL-18Rs, but not IL-1R, were expressed in DN1 and DN2 cells. (C) Flow cytometric analysis for the expression of IL-18R{alpha} in DN1 and DN2 cells.

 
A role of IL-18 for the development of CD11b+ DCs in the fetal thymus
To address the role of IL-18 in CD11b+ DC development, DN1 and DN2 cells were cultured in the presence of IL-18, IL-7 and SCF. Similar to the results obtained using ORTEC-CM, IL-18 induced the development of CD11c+CD11b+ cells as well as CD11c+I-Ab+ cells (Fig. 9A and Supplementary Figure 1, available at International Immunology Online). We also separated IL-18R{alpha}+ cells from E14.5 thymocytes and found that CD11c+CD11b+ cells were generated from them, indicating that IL-18 directly induced CD11b+ DC development from fetal thymocytes (data not shown). Furthermore, IL-18-induced DCs exhibited stronger APC activity than splenic DCs (Fig. 9B). These results indicate that IL-18 induces the differentiation of DN cells into functional DCs. These results suggest that IL-18 is involved in the development of CD11b+ DCs through the TRAF6 signaling pathway in vitro and in vivo.


Figure 9
View larger version (29K):
[in this window]
[in a new window]
  		Fig. 9 Role of IL-18 for DC differentiation and functions in the fetal thymus. (A) IL-18 induces the DC population from CD4–CD8– thymocytes. DN1 and DN2 cells were freshly isolated from E14.5 thymus and cultured for 6 days in the presence of IL-7 and SCF with IL-18. Cultured cells were stained with antibodies against CD11c, I-Ab and CD11b. IL-18 directly induced the development of DCs (CD11c+CD11b+ I-Ab+). (B) APC activity of the CD11c+I-Ab+ cells induced by IL-18 (closed squares) and splenic DCs (open circles) evaluated using the MLR assay. IL-18-induced DCs stimulated proliferation of allogenic CD4+ T cells. (C) Binding of DCs to DPT cells. IL-18-induced DCs or IL-1ß-induced DCs were co-cultured for 15 h with CD4+CD8{alpha}+ T (DPT) cells isolated from E16.5 thymi, at a DC:T cell ratio of 1:2. DPT cell control or DC control cells were cultured without mixing. Cultured cells were recovered and analyzed by flow cytometry. Three populations were recognized, i.e. CD3+CD11c– (T cell alone), CD3–CD11c+ (DC alone) and CD3+CD11c+ (T/DC conjugate). DPT cells formed more conjugates with IL-18-induced DCs (24%) than with IL-1ß-induced DCs (11%). (D) Binding potential of DCs with DPT. The fraction of CD11c+ cells that formed CD3+CD11c+ T/DC conjugates in all viable CD11c+ cells is shown. The result indicates that IL-18-induced DCs as well as thymic CD11b+ DCs exhibit greater potential to bind DPT than IL-1ß-induced DCs and thymic CD11b– DCs. Results are representative of two independent experiments. All experiments were performed in triplicate and error bars indicate standard deviation. *P < 0.02, **P > 0.1.

 
DCs are known to physically interact with T cells and the conjugation between T cells and DCs can be detected by flow cytometry (39). To investigate whether IL-18-induced DCs contribute to T cell selection in the thymus, we applied the T/DC conjugation assay to CD4+CD8+ (DPT) cells and IL-18-induced DCs to test whether they formed such conjugates, indicative of the antigen presentation to T cells. DPT cells (CD3+) were incubated with DCs at a DC:T ratio of 1:2 (10 000:20 000 per well). After 15 h of incubation, we analyzed the cells by flow cytometry and detected three populations, CD3+CD11c– (T cells), CD3–CD11c+ (DCs) and CD3+CD11c+ (T/DC conjugates) (Fig. 9C). When DPT cells were incubated with IL-18-induced DCs, 24% of all viable cells were in the T/DC conjugates. In sharp contrast, although the DCs developed from CD4–CD8– thymocytes upon treatment with the combination of IL-1ß, IL-3 and TNF-{alpha} as reported previously (19), those DCs (IL-1ß-induced DCs) formed T/DC conjugates with a lower extent (11%) than IL-18-induced DCs (Fig. 9C). The APC activity of IL-1ß-induced DCs was much weaker than that of ORTEC-CM-induced DCs or IL-18-induced DCs (data not shown), suggesting a functional difference between IL-18-induced DCs and IL-1ß-induced DCs. In contrast to the DPT cells, IL-18-induced DCs did not form T/DC conjugates with mature splenic CD4+ T (SPT) cells, thymic CD4+ T cells and CD8+ T cells (data not shown), suggesting that IL-18-induced DCs selectively recognize immature DPT cells but not mature SPT cells. The potential of DCs to form a conjugate with T cells indicate that IL-18-induced DCs bind DPT cells more efficiently than IL-1ß-induced DCs (Fig. 9D). In addition, CD11b+ DCs from the fetal thymus exhibited a much higher potential to interact with DPT than CD11b– DCs (Fig. 9D). These results strongly suggest that CD11b+ DCs play a major role for T cell development and that IL-18 is involved in the development of functional CD11b+ DCs in the fetal thymus.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
T cell development starts in the fetal thymus and immature thymocytes, the major population in the E16.5 thymus, differentiate into mature T cells via several steps. T cell development in the thymus is most active from fetal to perinatal stages and declines along with aging. A vast majority of T cells are eliminated by positive and negative selection in the thymus and thymic DCs are known to be involved in the establishment of self-tolerance (4, 24). However, the nature, origin and function of thymic DCs still remain unclear. We have found a novel class of DCs (i.e. CD8{alpha}–CD11b+ DCs) in the fetal thymus, which require TECs for development and may play a vital role in T cell selection. The CD11b+ DCs exhibit potent APC activity and bind DPT cells more efficiently than CD11b– DCs in the fetus (Figs 2C and 9C and D). The number of CD11b+ DCs is significantly reduced in TRAF6-deficient fetal thymus (Fig. 3). In consistent with this result, IL-18 produced by TECs induces the development of CD11b+ DCs from CD4–CD8– thymocytes (Fig. 9A).

Two classes of DCs had been recognized, i.e. DCs in the adult thymus are mostly CD8{alpha}+CD11b–, whereas DCs in secondary lymphoid tissues including spleen and lymph nodes are mainly CD8{alpha}–CD11b+ (5, 40). While CD8{alpha}+CD11b– DCs are the major class of DCs in the adult thymus, Dakic et al. (16) recently reported that DCs in the fetal thymus are negative for CD8{alpha}. We confirmed their result and further divided this population into CD11b+ and CD11b– (Fig. 1A). The CD8{alpha}–CD11b+ population declines after birth and becomes a minor population in the adult thymus (Fig. 1B). The period in which CD11b+ DCs are abundant in the thymus coincides with the period when T cell selection occurs most actively, suggesting that CD11b+ DCs may play a vital role in the T cell selection. In fact, CD11b+ DCs exhibit stronger APC activity in MLR assays and bind DPT cells more efficiently than CD11b– DCs from the fetal thymus. These results strongly suggest that CD11b+ cells are the major APCs for the DPT cells in the fetal thymus. CD11b+ DCs are reduced in TRAF6-deficient thymus (Fig. 3B) and TRAF6 deficiency in the thymus causes symptoms of autoimmunity (41). Thus, CD11b+ DCs may be involved in T cell selection as discussed below.

Thymic DCs are derived from intra-thymic precursors and we found that DN1 and DN2 cells give rise to CD11b+ DCs. As DN cells are a heterogeneous population, the exact origin of thymic DCs remains unknown. It was reported that thymic DCs were derived from T/DC precursors, while some DCs or their immediate precursors were reported to derive from the bloodstream in the adult thymus (23). Thus, whether thymic DCs derive from T cell precursors or myeloid precursors in the thymus has been a controversial issue. We show in this paper that the development of CD11b+ DCs from DN cells is induced by IL-18. The IL-18R was expressed in a significant fraction of DN1 and DN2 cells, in which the CD11c population was excluded to avoid a DC contamination for RT–PCR analysis. In addition, IL-7 is known to be required for survival and proliferation of lymphoid progenitors and we found that IL-18 strongly induced CD11b+ DCs in the presence of IL-7 (data not shown). Although we did not find evidence for TCRß rearrangement in the CD11b+ DCs (data not shown), it was reported that thymic DCs are derived from intra-thymic precursor at a stage before TCRß rearrangement (20). Thus, while it remains possible that CD11b+ DCs derive from a minor population of myeloid precursors with the IL-18Rs in the fetal thymus, it is strongly suggested that IL-18 produced from TECs induces development of DCs from intra-thymic T cell progenitors and that the IL-18R is an excellent marker for CD11b+ DC progenitors in the fetal thymus.

TEC is known to play a role in the positive selection of T cells by expressing MHC (42–44) and we recently showed that TECs play an active role in the development of thymic CD4+ macrophages from T cell progenitors (28, 29). We now show that TECs produce a factor, IL-18, which induces the differentiation of DCs from DN cells, suggesting that TECs play crucial roles in the T cell development by producing APCs (DCs) for T cell selection and scavengers (CD4+ macrophages) for the clearance of apoptotic T cells in the thymus. Consistently, it was recently shown that splenic stroma cells play a crucial role for the maturation of DCs in the spleen (45). Thus, microenvironment is an important determinant of the DC fate.

It was recently reported that CD4+CD11b+ splenic DCs, which exhibited characteristics of myeloid DCs, are nearly absent in TRAF6-deficient mice and TRAF6-deficient DCs are unable to up-regulate the surface expression of I-Ab, CD86 and inflammatory cytokines in response to microbial components, indicating an essential role for TRAF6 in the development and maturation of DCs in the adult spleen (32). In the 14-day-old mice, although hypoplasia of medullary TECs and reduced number of thymocytes were observed in TRAF6-deficient thymus, maturation and the absolute number of T cells were not significantly affected (41). However, in the neonatal thymus, the size and absolute cell numbers of thymi were not significantly different between WT and TRAF6-deficient mice (Akiyama et al., unpublished observation). Taken together, it is suggested that the absolute number of thymocytes in the E16.5 thymi is not significantly different between WT and TRAF6-deficient mice. We found a significant reduction of thymic CD11b+ DCs in the E16.5 thymus of TRAF6-deficient mice, indicating that a cytokine acting on the TRAF6 signaling pathway is required for the development of DCs in the fetal thymus in vivo. Among the cytokines known to activate TRAF6, only IL-18 was found to be abundantly expressed in TECs (Fig. 7A–C). While IL-18 production has been shown in macrophages, DCs, keratinocytes, osteoblasts and various epithelial cells (46, 47), in our knowledge this is the first report that IL-18 is produced by TECs. IL-18 expression in the thymus increases along with development (Fig. 7D) and the IL-18R is expressed on DN cells. Most significantly, IL-18 directly induces the differentiation of DN1 and DN2 cells into CD11b+ DCs (Fig. 9A). In contrast to fetal thymus, the fraction of CD11b+ DCs is very low in the adult thymus (Fig. 1A and B) and IL-18 failed to induce the development of CD11b+ DCs from adult CD4–CD8– thymocytes (data not shown). Although IL-18 expression was observed in the adult thymus (Fig. 7D), IL-18R expression in adult CD4–CD8– thymocytes was low; in particular, expression of IL-18Rß, which is important for signal transduction, was undetectable. These results indicate that IL-18 does not induce CD11b+ DCs in the adult thymus due to the lack of functional IL-18R in adult CD4–CD8– thymocytes. On the other hand, we found that the combination of IL-1ß, IL-3 and TNF-{alpha} induced the development of CD11b+ DCs from adult CD4–CD8– thymocytes as previously reported (19).

To address the function of IL-18 for fetal thymic DC development in vivo, we assessed CD11b+ DC development in IL-18R{alpha}-deficient fetal thymus (48) and found that the development of CD11b+ DCs was not severely affected in IL-18R{alpha}-deficient fetal thymus (Ito et al., unpublished observation). In consistent with this observation, IL-18 antagonists, i.e. IL-18-binding protein and neutralizing anti-IL-18Rß antibody, failed to inhibit the development of CD11b+ DCs from fetal thymocytes in the presence of ORTEC-CM. These results indicate the presence of another cytokine with the ability to induce CD11b+ DC via the TRAF6 signaling pathway in the fetal thymus. Because cytokines in the IL-1 family as well as the TNF-{alpha} family utilize the TRAF6 signaling pathway and there are still several members in both families which have not been well characterized (31, 49, 50), such cytokines may be a good candidate. Furthermore, novel factors that activate TRAF6 may be present in the fetal thymus and support CD11b+ DC development. In any cases, CD11b+ DCs generated by IL-18 in vitro show strong APC activity and the ability of DPT recognition in a manner similar to the CD11b+ DCs freshly isolated from the fetal thymus (Fig. 9). It is thus tempting to speculate that the CD11b+ DCs play a vital role for T cell selection by inducing apoptosis of DPT in the conjugates between DPT and DCs. Although DCs can be derived in vitro from thymocytes by the combination of cytokines, IL-1ß, IL-3, TNF-{alpha}, IL-7 and SCF, the APC activity as well as binding of those DCs to DPT cells is much weaker than that of IL-18-induced DCs. Thus, it is likely that the thymic CD11b+ DCs are novel DCs involved in the T cell selection during the perinatal stage. As TRAF6-deficient thymus develops autoimmune disease (41), the impaired development of CD11b+ DCs in the thymus may be one reason for the development of autoimmunity. A detailed analysis of the interaction between CD11c+CD11b+ cells and DPT cells will provide key information as to the molecular mechanism of T cell selection in the thymus and may also provide a molecular basis for the development of autoimmunity.

Among the B7 family, CD80 and CD86 on DCs are known to be involved in T cell stimulation via CD28 and CTLA-4 on T cells. Although B7 family members are important for peripheral immune responses, the role of CD80 and CD86 in negative selection still remains unclear, e.g. mice devoid of CD80 or CD86 show a little defect in T cell selection (51). While we found a high level of CD86 in CD11b+ thymic DCs, its role in DC functions remains to be investigated. A major obstacle to the analysis of thymic DCs has been that they are a very rare population. In this study, we have successfully developed a means to produce a large quantity of functional thymic DCs from immature thymocytes in vitro. Studies using these DCs will provide valuable information on the function of thymic DCs in vivo and also roles of co-stimulatory molecules in T cell selection.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data are available at International Immunology Online.


   Acknowledgements
 
We thank Drs Y. Kamogawa, K. Sekine, M.Tanaka, T. Itoh and M. Takeuchi for helpful suggestions and A. Hirata, T. Sekiguchi and Y. Ando for their excellent cell sorting. We are also very grateful to Drs S. Akira and K. Nakanishi for providing us with mutant mouse. This work was supported in part by Grants-in-Aid for Scientific Research and Special Coordination Funds from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from CREST program of JST.


   Abbreviations
 
7-AAD, 7-amino-actinomycin D
APC, antigen-presenting cell
CM, condition medium
DC, dendritic cell
DN, double negative
DPT, double positive T
E, embryonic day
FBS, fetal bovine serum
FTOC, fetal thymus organ culture
IL-18R{alpha}, IL-18R {alpha} chain
MLR, mixed lymphocytes reaction
ORTEC, OSM-responsive TEC line
OSM, oncostatin M
rm, recombinant murine
RT, reverse transcription
SCF, stem cell factor
SPT, single positive T
TEC, thymic epithelial cell
TNF, tumor necrosis factor
TRAF6, TNFR-associated factor 6
WT, wild type

   Notes
 
Transmitting editor: S. Koyasu

Received 4 January 2006, accepted 9 May 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

  1. Banchereau J and Steinman RM. (1998) Dendritic cells and the control of immunity. Nature 392:245.[CrossRef][Medline]
  2. Hart DN. (1997) Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90:3245.[Free Full Text]
  3. Banchereau J, Briere F, Caux C, et al. (2000) Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767.[CrossRef][Web of Science][Medline]
  4. Brocker T, Riedinger M, Karjalainen K. (1997) Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo. J. Exp. Med. 185:541.[Abstract/Free Full Text]
  5. Shortman K and Liu YJ. (2002) Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2:151.[CrossRef][Web of Science][Medline]
  6. Ardavin C. (2003) Origin, precursors and differentiation of mouse dendritic cells. Nat. Rev. Immunol. 3:582.[CrossRef][Web of Science][Medline]
  7. Aliberti J, Schulz O, Pennington DJ, et al. (2003) Essential role for ICSBP in the in vivo development of murine CD8alpha + dendritic cells. Blood 101:305.[Abstract/Free Full Text]
  8. Guerriero A, Langmuir PB, Spain LM, Scott EW. (2000) PU.1 is required for myeloid-derived but not lymphoid-derived dendritic cells. Blood 95:879.[Abstract/Free Full Text]
  9. O'Keeffe M, Hochrein H, Vremec D, et al. (2002) Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte-macrophage colony-stimulating factor on dendritic cell subsets in mice. Blood 99:2122.[Abstract/Free Full Text]
  10. Wu L, D'Amico A, Winkel KD, Suter M, Lo D, Shortman K. (1998) RelB is essential for the development of myeloid-related CD8alpha– dendritic cells but not of lymphoid-related CD8alpha+ dendritic cells. Immunity 9:839.[CrossRef][Web of Science][Medline]
  11. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K. (1997) Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 7:483.[CrossRef][Web of Science][Medline]
  12. Suzuki S, Honma K, Matsuyama T. (2004) Critical roles of interferon regulatory factor 4 in CD11bhighCD8alpha- dendritic cell development. Proc. Natl Acad. Sci. USA 101:8981.[Abstract/Free Full Text]
  13. Vremec D, Zorbas M, Scollay R, et al. (1992) The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J. Exp. Med. 176:47.[Abstract/Free Full Text]
  14. Vremec D and Shortman K. (1997) Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J. Immunol. 159:565.[Abstract]
  15. Vremec D, Pooley J, Hochrein H, Wu L, Shortman K. (2000) CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. Immunol 164:2978.[Abstract/Free Full Text]
  16. Dakic A, Shao QX, D'Amico A, et al. (2004) Development of the dendritic cell system during mouse ontogeny. J. Immunol. 172:1018.[Abstract/Free Full Text]
  17. Moore TA and Zlotnik A. (1995) T-cell lineage commitment and cytokine responses of thymic progenitors. Blood 86:1850.[Abstract/Free Full Text]
  18. Wu L, Li CL, Shortman K. (1996) Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. J. Exp. Med. 184:903.[Abstract/Free Full Text]
  19. Saunders D, Lucas K, Ismaili J, et al. (1996) Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 184:2185.[Abstract/Free Full Text]
  20. Shen HQ, Lu M, Ikawa T, et al. (2003) T/NK bipotent progenitors in the thymus retain the potential to generate dendritic cells. J. Immunol. 171:3401.[Abstract/Free Full Text]
  21. Radtke F, Ferrero I, Wilson A, Lees R, Aguet M, MacDonald HR. (2000) Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells. J. Exp. Med. 191:1085.[Abstract/Free Full Text]
  22. Rodewald HR, Brocker T, Haller C. (1999) Developmental dissociation of thymic dendritic cell and thymocyte lineages revealed in growth factor receptor mutant mice. Proc. Natl Acad. Sci. USA 96:15068.[Abstract/Free Full Text]
  23. Donskoy E and Goldschneider I. (2003) Two developmentally distinct populations of dendritic cells inhabit the adult mouse thymus: demonstration by differential importation of hematogenous precursors under steady state conditions. J. Immunol. 170:3514.[Abstract/Free Full Text]
  24. Suss G and Shortman K. (1996) A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J. Exp. Med. 183:1789.[Abstract/Free Full Text]
  25. Reis e Sousa C. (2001) Dendritic cells as sensors of infection. Immunity 14:495.[CrossRef][Web of Science][Medline]
  26. Naito A, Azuma S, Tanaka S, et al. (1999) Severe osteopetrosis, defective interleukin-1 signalling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4:353.[Abstract]
  27. Klein L, Klugmann M, Nave KA, Tuohy VK, Kyewski B. (2000) Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells. Nat. Med. 6:56.[CrossRef][Web of Science][Medline]
  28. Esashi E, Ito H, Ishihara K, Hirano T, Koyasu S, Miyajima A. (2004) Development of CD4+ macrophages from intrathymic T cell progenitors is induced by thymic epithelial cells. J. Immunol. 173:4360.[Abstract/Free Full Text]
  29. Esashi E, Sekiguchi T, Ito H, Koyasu S, Miyajima A. (2003) Cutting edge: a possible role for CD4+ thymic macrophages as professional scavengers of apoptotic thymocytes. J. Immunol. 171:2773.[Abstract/Free Full Text]
  30. Szilvassy SJ, Weller KP, Lin W, et al. (1996) Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells. Blood 87:4618.[Abstract/Free Full Text]
  31. Wu H and Arron JR. (2003) TRAF6, a molecular bridge spanning adaptive immunity, innate immunity and osteoimmunology. Bioessays 25:1096.[CrossRef][Web of Science][Medline]
  32. Kobayashi T, Walsh PT, Walsh MC, et al. (2003) TRAF6 is a critical factor for dendritic cell maturation and development. Immunity 19:353.[CrossRef][Web of Science][Medline]
  33. Inaba K, Inaba M, Romani N, et al. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176:1693.[Abstract/Free Full Text]
  34. Inaba K, Inaba M, Deguchi M, et al. (1993) Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow. Proc. Natl Acad. Sci. USA 90:3038.[Abstract/Free Full Text]
  35. Maraskovsky E, Brasel K, Teepe M, et al. (1996) Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184:1953.[Abstract/Free Full Text]
  36. Fukao T, Matsuda S, Koyasu S. (2000) Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-gamma production by dendritic cells. J. Immunol. 164:64.[Abstract/Free Full Text]
  37. Dinarello CA. (1998) Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int. Rev. Immunol. 16:457.[Medline]
  38. Dayer JM. (2002) Evidence for the biological modulation of IL-1 activity: the role of IL-1Ra. Clin. Exp. Rheumatol. 20:S14.[Web of Science][Medline]
  39. Watanabe N, Hanabuchi S, Soumelis V, et al. (2004) Human thymic stromal lymphopoietin promotes dendritic cell-mediated CD4+ T cell homeostatic expansion. Nat. Immunol. 5:426.[CrossRef][Web of Science][Medline]
  40. Wu L, D'Amico A, Hochrein H, O'Keeffe M, Shortman K, Lucas K. (2001) Development of thymic and splenic dendritic cell populations from different hemopoietic precursors. Blood 98:3376.[Abstract/Free Full Text]
  41. Akiyama T, Maeda S, Yamane S, et al. (2005) Dependence of self-tolerance on TRAF6-directed development of thymic stroma. Science 308:248.[Abstract/Free Full Text]
  42. Gutierrez JC and Palacios R. (1991) Heterogeneity of thymic epithelial cells in promoting T-lymphocyte differentiation in vivo. Proc. Natl Acad. Sci. USA 88:642.[Abstract/Free Full Text]
  43. Saunders DJ, Georgiou HM, Wu L, Shortman K. (1995) Induction of limited growth and differentiation of early thymic precursor cells by thymic epithelial cell lines. Immunol. Lett. 47:45.[CrossRef][Web of Science][Medline]
  44. Nehls M, Kyewski B, Messerle M, et al. (1996) Two genetically separable steps in the differentiation of thymic epithelium. Science 272:886.[Abstract]
  45. Zhang M, Tang H, Guo Z, et al. (2004) Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells. Nat. Immunol. 5:1124.[CrossRef][Web of Science][Medline]
  46. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. (2001) Interleukin-18 regulates both Th1 and Th2 responses. Annu. Rev. Immunol. 19:423.[CrossRef][Web of Science][Medline]
  47. Okazawa A, Kanai T, Nakamaru K, et al. (2004) Human intestinal epithelial cell-derived interleukin (IL)-18, along with IL-2, IL-7 and IL-15, is a potent synergistic factor for the proliferation of intraepithelial lymphocytes. Clin. Exp. Immunol. 136:269.[CrossRef][Web of Science][Medline]
  48. Hoshino K, Tsutsui H, Kawai T, et al. (1999) Cutting edge: generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor. J. Immunol. 162:5041.[Abstract/Free Full Text]
  49. Grant EP, Coyle AJ, Gutierrez-Ramos JC. (2003) Informatics and the immune system: the expanding IL-1 and B7 protein families. Semin. Immunol. 15:225.[CrossRef][Web of Science][Medline]
  50. Dunn E, Sims JE, Nicklin MJ, O'Neill LA. (2001) Annotating genes with potential roles in the immune system: six new members of the IL-1 family. Trends Immunol. 22:533.[CrossRef][Web of Science][Medline]
  51. Buhlmann JE, Elkin SK, Sharpe AH. (2003) A role for the B7-1/B7-2:CD28/CTLA-4 pathway during negative selection. J. Immunol. 170:5421.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow Supplementary Data
Right arrow All Versions of this Article:
18/8/1253    most recent
dxl058v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ito, H.
Right arrow Articles by Miyajima, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ito, H.
Right arrow Articles by Miyajima, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?