Skip Navigation



International Immunology Advance Access published online on January 9, 2008
International Immunology, doi:10.1093/intimm/dxm143
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrowOA All Versions of this Article:
20/3/307    most recent
dxm143v1
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Google Scholar
Right arrow Articles by Guo, Z.
Right arrow Articles by Miyasaka, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guo, Z.
Right arrow Articles by Miyasaka, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press on behalf of The Japanese Society for Immunology. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press and The Japanese Society for Immunology are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

CD4+CD25+ regulatory T cells in the small intestinal lamina propria show an effector/memory phenotype

Zijin Guo1,*, Myoung Ho Jang1,2,*, Kazuhiro Otani1, Zhongbin Bai1, Eiji Umemoto1, Masanori Matsumoto1,3, Mika Nishiyama1, Mikako Yamasaki1, Satoshi Ueha4, Kouji Matsushima4, Takako Hirata3 and Masayuki Miyasaka1,5

1 Laboratory of Immunodynamics, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
2 Laboratory of Gastrointestinal Immunology, WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan
3 The 21st Century Center of Excellence Program, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
4 Department of Molecular Preventive Medicine and SORST, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
5 Laboratory of Immunodynamics, WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan

Correspondence to: Correspondence to: M. Miyasaka; E-mail: mmiyasak{at}orgctl.med.osaka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
CD4+CD25+ regulatory T cells (Tregs) have been implicated in the suppression of pathogenic responses to both self- and non-self-antigens in the intestine. However, their precise properties and functions in the gut, as well as the molecular basis of their recruitment to the gut, are poorly understood. Here, we found that most of the CD4+CD25+ T cells in the small intestinal lamina propria (LP) express Foxp3 and exhibit an ‘effector/memory’ phenotype, CD44hiCD45RBloCD62L–, whereas only a minority of the Foxp3+CD4+CD25+ T cells in the spleen and mesenteric lymph nodes showed this phenotype. The Tregs in the small intestinal LP (LP-Tregs) expressed higher levels of CCR4 and CCR9 and a substantially lower level of CCR7 than the Tregs in the spleen. In vitro, the LP-Tregs showed chemotaxis to CCL25/thymus-expressed chemokine. In addition, they showed efficient chemotaxis to the CCR4 ligands, CCL17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine, which are abundantly expressed by dendritic cells (DCs) in the small intestinal LP. In vivo, ~50% of the LP-Tregs were closely associated or in direct contact with LP-DCs. These findings demonstrate that LP-Tregs are phenotypically and functionally unique and raise the possibility that they are retained in the small intestinal LP through the action of CCL17 and CCL22, which are locally produced by LP-DCs.

Keywords: cell migration, chemokines, dendritic cells, mucosal immunity, regulatory T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
CD4+CD25+Foxp3+ regulatory T cells (Tregs) are critical for immunological tolerance and they control various immune responses associated with allergy, infection and tumor immunity by patrolling the secondary lymphoid organs and peripheral tissues (1, 2). To traffic to these tissues, Tregs appear to depend on chemokine–chemokine receptor interactions (3). For example, Tregs migrate to the bone marrow partly through CXCL12–CXCR4 interactions (4). Tregs in cardiac allograft (5) and ovarian tumor (6) utilize CCR4 signals for their tissue localization. CD103– Tregs in the spleen and lymph nodes express CCR7 and preferentially migrate into lymph nodes, where CCR7 ligands are abundant, whereas CD103+ Tregs express receptors for inflammatory chemokines and preferentially migrate into inflamed sites (7, 8).

Tregs are also found in the intestine (9, 10). Whereas T cells and IgA plasma cells migrate to the intestine via the interaction between CCR9 and its ligand, CCL25/thymus-expressed chemokine (TECK) (11–14) [CCL25 is produced by intestinal epithelial cells (11) and presented on the intestinal venular endothelial cells (12)], it remains unclear whether Tregs in the intestine express functional CCR9. Furthermore, it is unknown how Tregs migrate into the gut and whether the mechanism they use is distinct from that regulating the Treg migration to other tissues.

Tregs are self-reactive. Therefore, interactions with self-antigens are probably essential for their development and function (15–20). Observations by us (21) and others (22) indicate that such interactions occur in the intestinal mucosa; distinct dendritic cell (DC) subsets of the small intestinal lamina propria (LP) constitutively endocytose self-antigens derived from apoptotic intestinal epithelial cells and transport them to the T-cell areas of mesenteric lymph nodes (MLNs), where Tregs are localized. Given that DCs can present self-antigens and imprint tissue tropism on T cells, it is tempting to speculate that these DC subsets can also prime Tregs locally and affect their imprinting mechanisms to confer tissue tropism to the LP and MLNs on the Tregs.

In this study, we found that a substantial proportion of the CD4+CD25+ cells in the small intestinal LP expressed Foxp3 and showed an ‘effector/memory’ phenotype, CD44hiCD45RBlo. Furthermore, the Tregs in the LP (LP-Tregs) expressed high levels of CCR4 and showed efficient chemotaxis to the CCR4 ligands CCL17/thymus and activation-regulated chemokine (TARC) and CCL22/macrophage-derived chemokine (MDC). We also found that CCL17 and CCL22 were selectively expressed by DCs in the small intestinal LP and that Tregs were often found in close proximity to these DCs in the LP. These findings are consistent with the hypothesis that Tregs localize to the small intestinal LP through the action of CCL17 and CCL22 produced by LP-DCs and that the DCs influence the Tregs' activities locally.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Animals
BALB/c mice (5–8 weeks old) were purchased from CLEA Japan. All animal experiments were performed under protocols approved by the Ethics Review Committee for Animal Experimentation of Osaka University Medical School.

Antibodies and chemokines
Purified anti-CD16/CD32 (2.4G2), purified anti-CD3 (145-2C11), purified anti-CD28 (37.51), purified and biotinylated anti-CD11c (HL3), FITC, PE–Cy5-conjugated and biotinylated anti-CD4 (RM4-5), PE-conjugated anti-integrin β7 (M293), FITC-conjugated anti-CD62L (MEL14) mAbs, PE-conjugated anti-CTLA-4 (UC10-4F10-11) and APC-conjugated streptavidin were purchased from BD PharMingen (San Jose, CA, USA). PE-conjugated anti-CD25 (PC61.5) and APC-conjugated anti-Foxp3 (FJK-16s) mAbs were from eBioscience (San Diego, CA, USA). Purified goat anti-mouse CCR4 pAb was from Capralogics (Hardwick, MA, USA). Anti-mouse CCR9 mAb (7E7) was kindly provided by Reinhold Foerster (Hannover Medical School, Germany). Rabbit anti-mouse Foxp3 pAb was prepared as described previously (23). CCL19–Fc, CXCL12–Fc chimeric proteins and human IgG Fc were kindly provided by K. Hieshima and O. Yoshie (Kinki University School of Medicine, Japan). Biotin-conjugated goat anti-human IgG pAb was from American Qualex (San Clemente, CA, USA). Alexa 647- and Alexa 594-conjugated streptavidin, Alexa 488- and Alexa 647-conjugated chicken anti-goat IgG pAb and 4',6-diamidino-2-phenylindole were purchased from Molecular Probes (Eugene, OR, USA). Recombinant mouse CCL25, CCL17 and CCL22, goat anti-mouse CCL17 pAb and rat anti-mouse CCL22 mAb were purchased from R&D Systems (Minneapolis, MN, USA).

Preparation of CD4+CD25+ T cells from small intestinal LP, MLNs and spleen
The small intestine was opened longitudinally along the mesenteric wall, and the Peyer's patches and isolated lymphoid follicles were removed as described previously (24). Small intestinal segments were treated with PBS containing 10% FCS, 20 mM HEPES, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin, 1 mM sodium pyruvate, 10 mM EDTA and 10 µg ml–1 polymyxin B (Calbiochem, San Diego, CA, USA) for 30 min at 37°C to remove epithelial cells and were then washed extensively with PBS. The segments were then treated with 400 Mandl U ml–1 collagenase D (Roche, Penzberg, Germany) and 100 µg ml–1 DNase I (Roche) in RPMI 1640/10% FCS with continuous stirring at 37°C for 30–45 min. EDTA was added (10 mM final concentration), and the cell suspension was incubated for an additional 5 min at 37°C. After washing, the cells were subjected to density gradient centrifugation through 45%/75% Percoll (approximate density 1.058 and 1.093 g ml–1, respectively). The cells were harvested from the interface, washed and used for assays.

A single-cell suspension was prepared from the spleen or MLNs by mincing the tissue on a 100-µm pore-size cell strainer (BD Falcon, San Jose, CA, USA). RBCs were removed by treating the cells with ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3 and 0.1 mM Na2EDTA, pH 7.2). For FACS sorting, the cells were stained with PE-conjugated anti-CD25 and PE–Cy5-conjugated anti-CD4 mAbs. CD4+CD25+ T cells were sorted using a FACSVantage SE (BD Biosciences, San Jose, CA, USA). The purity of the sorted CD4+CD25+ T cells was routinely >95%. For the positive selection of CD25+ cells, the cell suspension was incubated with PE-conjugated anti-CD25 and then with anti-PE beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and subjected to AutoMACS (Miltenyi Biotec).

Flow cytometry
Freshly isolated cells were incubated with Fc BlockTM for 15 min at 4°C and then stained for CD4 and CD25. The intracellular staining of Foxp3 was conducted using the protocol recommended by BD PharMingen. The cells were analyzed on a FACSCalibur (BD Biosciences). Tregs were identified by gating on the CD4+CD25+ cell population. The expression of CCR4 was determined using anti-CCR4 pAb and Alexa 647-conjugated chicken anti-goat IgG pAb. CCR9 expression was determined with an anti-CCR9 mAb, which we labeled with Alexa 647. CCR7 and CXCR4 expression was determined using the CCL19–Fc and CXCL12–Fc chimeric proteins, respectively. The Fc domain was then detected by biotinylated goat anti-human IgG pAb followed by incubation with APC-conjugated streptavidin.

Immunohistochemistry
To detect Tregs, frozen sections were fixed in methanol at –30°C for 10 min, and then rabbit anti-mouse Foxp3 pAb and biotinylated anti-CD4 mAb were applied overnight at 4°C. Samples were washed and then incubated with Alexa 594-conjugated streptavidin and Alexa 488-conjugated chicken anti-rabbit IgG pAb for 2 h at room temperature (RT). To detect DCs, sections were incubated with biotinylated anti-CD11c mAb overnight at 4°C followed by incubation with Alexa 594-conjugated streptavidin for 1.5–2 h at RT. Immunohistochemical staining was analyzed using a Radiance 2100/Bio-Rad confocal laser microscope (Bio-Rad, Hemel Hempstead, UK).

Quantitative real-time PCR
Total RNA was prepared from freshly isolated cells using TRIzol RNA extraction reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was prepared using WT-Ovation RNA Amplification System (NuGEN Technologies, Inc., San Carlos, CA, USA), according to the manufacturer's instructions. Quantitative real-time PCR was conducted in a final volume of 25 µl containing amplified cDNA and 2x PCR Master Mix (Applied Biosystems), and the following inventoried primer sets [CCL17, CCL22 and CCR4 (Applied Biosystems)] were used. β-Actin was used as an internal control.

T-cell suppression assay
Sorted CD4+CD25– splenocytes were used as indicator cells and labeled with 5 µM CFSE. They were then mixed with one of the following: (i) LP-derived CD11chi cells plus LP CD4+CD25+ cells, (ii) LP-derived CD11chi cells plus LP CD4+CD25– cells, (iii) splenic CD11chi cells plus splenic CD4+CD25+ cells or (iv) splenic CD11chi cells plus splenic CD4+CD25– cells. These three cell types were mixed at a 1:1:1 ratio and were stimulated by culturing in the presence of 5 µg ml–1 anti-CD3 mAb and 2 µg ml–1 anti-CD28 mAb. After 3 days, the cellular proliferation was assessed by CFSE dilution using a FACSCalibur.

Transwell chemotaxis assay
Chemotaxis assays were performed using 5-µm pore polycarbonate filters in a Transwell chamber (Corning Costar Corporation) as described (21). CD25+ cells enriched by AutoMACS were cultured in complete RPMI10 with 8 ng ml–1 IL-2 (R&D Systems) for 1–2 h before being applied to the upper well. All the cell suspensions and chemokine dilutions were prepared in RPMI 1640 containing 0.5% low-endotoxin fatty acid-free BSA (Sigma–Aldrich) and 8 ng ml–1 IL-2. Conditioned medium was prepared from LP-DCs and splenic DCs. The LP-DCs and splenic DCs (2 x 105) were incubated in 3 ml culture medium in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF). After 24 h, the culture medium was collected and used in chemotaxis assay. Before applied to chemotaxis assay, culture medium was incubated with 10 µg ml–1 anti-CCL17 antibody and/or anti-CCL22 antibody or with rat Ig at 37°C for 30 min. After 2.5 h of incubation, the inserts were removed and 5 x 104 polystyrene beads (Polysciences, Warrington, PA, USA) were added to the lower compartment of the Transwell. The migrated cells were recovered and stained with PE–Cy5-conjugated anti-CD4 mAb and PE-conjugated anti-CD25 mAb. The number of migrated T cells was measured by the flow cytometer acquisition of a fixed number of beads. To calculate the specific migration, the number of migrated cells in the absence of chemokine was subtracted from the number of the corresponding cell sub-population that had migrated in the presence of chemokines. To evaluate the percentage of migration, the number of migrated CD4+CD25+ T cells was divided by the total number of input CD4+CD25+ T cells.

ELISA
LP-DCs, LP-eosinophils (EOs) and splenic DCs were isolated as described (21) and sorted by FACSVantage SE, after which the purity of each cell type was >98%. Samples of 6 x 105 cells were incubated in 1.5 ml cRPMI10. After 40 h, the medium was collected. The levels of TARC and MDC in the culture medium were measured with the Quantikine® system (R&D Systems), according to the manufacturer's protocol. The plates were read at 450 nm in a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The concentrations were calculated from a standard curve.

Statistical analysis
Data are presented as the mean ± SD, as indicated. The cells were isolated from three or four mice in each experiment. All experiments were repeated more than three times.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
The CD4+CD25+ T cells in the small intestinal LP express Foxp3
Intestinal CD4+CD25+ Tregs are likely to play pivotal roles in the induction and maintenance of intestinal tolerance to both self- and intestinal antigens locally. Although the large intestinal CD4+CD25+ Tregs have been partially characterized (9, 10), relatively little is known about small intestinal CD4+CD25+ Tregs, particularly in terms of their frequency, intraintestinal localization, phenotype and functional capacities. To examine the abundance of Tregs in the small intestine, we first determined the frequency of CD4+CD25+ T cells in the small intestinal LP and compared it with their frequency in other secondary lymphoid organs. To exclude the contribution of Tregs derived from the Peyer's patches and/or isolated lymphoid follicles, we used mice whose Peyer's patches and isolated lymphoid follicles had been surgically removed. As shown in Fig. 1(A), 10–15% of the CD4+ T cells were CD25+ in the small intestinal LP, spleen and MLNs, indicating that Tregs are present in the small intestinal LP and that their frequency among CD4+ cells is similar to that seen in other secondary lymphoid tissues.


Figure 1
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. CD4+CD25+ T-lymphocytes in the small intestinal LP express Foxp3. (A) Lymphocytes from the small intestinal LP, spleen (SP) and MLNs were stained for CD4 and CD25 and analyzed by flow cytometry. The FACS profiles were acquired by gating on CD4. (B) Cells were permeabilized and stained with a rat anti-mouse Foxp3 mAb; the histogram profiles were acquired by gating on CD4+CD25+ T cells or CD4+CD25– T cells. Gray histograms, cells incubated with isotype control IgG and open histograms, cells incubated with rat anti-mouse Foxp3 mAb. (C) CD4+CD25+ T cells and CD4+CD25– T cells from the small intestinal LP were FACS sorted and stained with rabbit anti-mouse Foxp3 pAb (green). The nuclei were then stained with Hoechst 33258 (blue). (D) Proportion of CD4+CD25+ or CD4+CD25– T cells that were Foxp3+ in the small intestinal LP and SP. Cells were counted in five non-consecutive microscopic fields and the mean ± SD was calculated. Data are representative of two independent experiments.

 
Because CD25 is a marker of cell activation and not necessarily an indicator of Tregs on its own, we next examined the expression of Foxp3, a bona fide marker for Tregs (1), in the CD4+CD25+ and CD4+CD25– cells derived from these tissues, by flow cytometry using an mAb against Foxp3. As shown in Fig. 1(B), a substantial proportion of CD4+CD25+ cells expressed Foxp3 in the small intestinal LP, MLNs and spleen, and strong Foxp3 expression was observed selectively in the CD4+CD25+ T cells. These results indicate that Tregs are abundant in the small intestinal LP, as in other lymphoid tissues, and are contained exclusively in the CD4+CD25+ cell population. In addition, Tregs in the LP do not appear to be derived from Peyer's patches or isolated lymphoid follicles. Supporting the flow cytometry data, immunocytochemical staining of CD4+CD25+ and CD4+CD25– cells from the small intestinal LP using polyclonal anti-Foxp3 antibodies showed that Foxp3 was expressed almost exclusively in the nucleus of the CD4+CD25+ cells (Fig. 1C). The majority of CD4+CD25+ cells in the LP, MLN and spleen was Foxp3+, whereas the CD4+CD25– cells in these tissues hardly expressed Foxp3 (Fig. 1D).

CD4+Foxp3+ cells are found in the T-cell area of MLNs and throughout the intestinal LP
We next examined the localization of CD4+Foxp3+ Tregs in the small intestinal LP and MLNs by immunohistochemistry, using the anti-Foxp3 pAb. In the small intestinal LP, CD4+Foxp3+ cells were readily recognizable throughout the LP region (Fig. 2A), although not as densely as in the spleen or MLNs (Fig. 2B and C). No non-specific staining was observed with control normal rabbit serum (data not shown). Considering the ample surface area of the small intestinal LP, however, these results indicate an overall abundance of Tregs in the small intestinal LP.


Figure 2
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. CD4+Foxp3+ Tregs are localized to the small intestinal LP. Frozen sections of the small intestine (A), spleen (SP) (B) and MLNs (C) were stained with antibodies specific for CD4 (red) and Foxp3 (green). Lymphoid follicles were determined by staining for B220 (blue) in the sections of SP and MLNs. The sections were analyzed by confocal microscopy. CD4+ T cells expressing Foxp3 were found within the villi and scattered throughout the LP (LP1 and LP2). In panel (A), epithelial cells are delineated by the dotted lines. Small intestine: original magnification, x400 (left) and x1200 (right). SP and MLN: original magnification, x400 and x1200 (inset panel).

 
Tregs in LP highly express CTLA-4 and possess suppressive function in vitro
We next examined the expression of CTLA-4 on CD4+Foxp3+ and CD4+Foxp3– cells in LP and spleen because CTLA-4 is critical for Treg function. As shown in Fig. 3(A), LP CD4+Foxp3+ cells expressed CTLA-4 at much higher levels than did splenic CD4+Foxp3+ cells, whereas LP CD4+Foxp3– cells expressed little or no CTLA-4.


Figure 3
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. CD4+CD25+ Tregs in the small intestinal LP show an effector/memory phenotype. (A) Lymphocytes from the spleen and LP were stained for CD4, Foxp3 and CTLA-4. FACS profiles were acquired by gating on the CD4+Foxp3+ cells. LP CD4+Foxp3+ T cells showed higher expression of CTLA-4 than splenic CD4+Foxp3+ T cells. (B) In vitro suppression assay. Purified splenic CD4+CD25– T cells were labeled with 5 µM CFSE and co-cultured with either of the following: (i) LP-derived CD11chi cells plus LP CD4+CD25+ cells, (ii) LP-derived CD11chi cells plus LP CD4+CD25– cells, (iii) splenic CD11chi cells plus splenic CD4+CD25+ cells or (iv) splenic CD11chi cells plus splenic CD4+CD25– cells. The CFSE-labeled cell, CD11chi cells and test cells were mixed at a ratio 1:1:1. After 3 days, CFSE dilution was analyzed by flow cytometry. (C) Lymphocytes from the spleen, MLNs and LP were stained for CD4, CD25, CD62L and β7 integrin. FACS profiles were acquired by gating on the CD4+CD25+ cells. Based on the integrin β7 and CD62L expression, three Treg subsets were recognized: CD62L–β7hi (I), CD62L–β7lo (II) and CD62L+β7lo (III). Bar graph showing the percentage of total Tregs represented by each subset. Note that the Tregs in spleen and MLNs mainly consisted of subset III, whereas the LP–Tregs were mostly of subset I or II. (D) Lymphocytes were stained for CD4, CD25, CD44 and CD45RB. FACS profiles were acquired by gating on the CD4+CD25+ cells. Based on the CD44 and CD45RB expression, the CD4+CD25+ cells were grouped into two sub-populations, CD44hiCD45RBlo and CD44intCD45RBhi. (E) CD103 expression on CD4+CD25+ cells: CD103+ cells are shown by the thick-line histogram. Control antibody staining is shown as the thin-line histogram.

 
To test the ability of LP-Tregs to suppress T-cell responses, we compared the ability of purified CD4+CD25+ cells from LP and spleen to inhibit proliferation of CD4+CD25– T cells in vitro. As shown in Fig. 3(B), although both LP and splenic CD4+CD25+ T cells suppressed T-cell proliferation, the LP CD4+CD25+ T cells exhibited substantially lower suppressive activity than the splenic CD4+CD25+ T cells. In contrast, neither LP CD4+CD25– cells nor splenic CD4+CD25– cells suppressed T-cell proliferation. These data may indicate intrinsically weaker regulatory activities of the LP CD4+CD25+ T cells, although we cannot rule out contamination of the LP CD4+CD25+ cell population by activated T cells at present.

The majority of Tregs in the LP shows down-regulated CD62L expression and bears an effector/memory phenotype, CD44hiCD45RBlo
We next examined the expression of CD62L together with that of another critical cell adhesion molecule, β7 integrin, on CD4+CD25+ cells in the intestinal LP, MLNs and spleen. We could recognize three phenotypically distinct subsets: CD62L–β7hi (subset I), CD62L–β7lo (subset II) and CD62L+β7lo (subset III) in these tissues (Fig. 3C). Interestingly, whereas the CD62L-expressing subset III was the most abundant in MLNs and spleen, this subset was only a minority in the small intestinal LP (Fig. 3C); thus, the majority of CD4+CD25+ LP T cells lacked CD62L expression. Immunohistochemical analysis confirmed that most of the Foxp3+ cells in the intestinal LP expressed little or no CD62L (data not shown). Upon antigen activation, T cells rapidly shed CD62L, a critical lymphocyte adhesion molecule for lymph node trafficking (25). Tregs also show CD62L shedding upon activation, although CD62L expression does not seem to correlate with their suppressive function in vitro (26). Our results thus indicate that LP-Tregs are phenotypically unique and that most of them show a down-regulated expression of CD62L. Furthermore, as shown in Fig. 3(D), LP-Tregs showed lower CD45RB expression than the splenic and MLN–Tregs; they also showed high CD44 expression. Thus, the majority (81.7%) of LP-Tregs bore an effector/memory phenotype, CD44hiCD45RBlo.

Previous studies have shown that CD103+-Tregs preferentially migrate into effector/inflammatory sites (7, 8). Although the frequency was higher than in spleen (19.4%) and MLNs (13.6%), only 25.7% of the LP-Tregs expressed CD103 (Fig. 3E), arguing against the possibility that CD103 is a critical homing molecule for Tregs that migrate into the small intestine under physiological conditions.

LP-Tregs highly express CCR4 and respond efficiently to the CCR4 ligands CCL17 and CCL22
Whereas the trafficking of gut-tropic T cells is regulated by CCR9-mediated signaling (11, 27, 28), the involvement of chemokines in Treg trafficking to the intestine remains to be established. To address this issue, we first investigated chemokine receptor expression in intestinal CD4+CD25+ Tregs. As shown in Fig. 4(A), LP-Tregs expressed substantially higher levels of CCR4 and CCR9, lower levels of CCR7 and comparable levels of CXCR4, compared with the CD4+CD25+ Tregs from the spleen. Consistently, quantitative real-time PCR analysis showed that the small intestinal LP-Tregs expressed much higher levels of CCR4 than did the CD4+CD25+ Tregs of other tissues (Fig. 4B).


Figure 4
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. LP-Tregs express chemokine receptor CCR4 and respond to CCR4 ligands. (A) Expression of CCR4, CCR7, CCR9 and CXCR4 on CD4+Foxp3+ cells of the small intestinal LP and spleen. Lymphocytes were stained for CCR4 and CCR9 with specific mAbs (open histograms) or isotype Ig controls (gray histograms). To detect CCR7 and CXCR4, the cells were stained by the CCL19–Fc and CXCL12–Fc chimeric proteins (open histograms) or with human IgG Fc (gray histograms). The histogram profiles were acquired by gating on the CD4+Foxp3+ cells. (B) Expression of CCR4 mRNA. The cDNA was prepared from total RNA obtained from freshly FACS-sorted CD4+CD25+ Tregs, and the expression of CCR4 was analyzed by quantitative real-time PCR. The CCR4 mRNA levels were normalized to β-actin and are shown as the expression relative to that of CD4+CD25+ Tregs from the spleen. (C) LP-Tregs show chemotaxis to CCR4 ligands and CCL25. Lymphocytes were isolated from the LP and spleen and 106 cells were added to the upper well of a Transwell chamber. After a 2.5- to 3-h incubation, the cells in the lower well were collected, stained for CD4 and CD25 and analyzed by flow cytometry. The bar graph shows the migratory responses of CD4+CD25+ T cells toward serial dilutions of CCL17, CCL22 and CCL25. The LP-Tregs responded to CCR4 ligands and CCL25, but the splenic Tregs responded only poorly.

 
The high CCR4 and CCR9 expression by intestinal LP CD4+CD25+ Tregs is apparently functionally relevant because LP-derived CD4+CD25+ cells showed efficient chemotaxis in response to the CCR4 ligands CCL17/TARC and CCL22/MDC (Fig. 4C), and this chemotaxis was completely abrogated by the addition of neutralizing antibodies against these chemokines (data not shown). LP-derived CD4+CD25+ cells also showed chemotaxis to CCL25/TECK (Fig. 4C). Intestinal LP CD4+CD25– T cells also showed efficient chemotaxis in response to CCL17 and CCL22, suggesting that CCR4-mediated chemotaxis is not selective to CD4+CD25+ T cells but rather general to intestinal LP CD4+ T cells (Fig. 4C). In contrast, splenic T cells (CD4+CD25+ and CD4+CD25–) showed chemotaxis to CCL17, CCL22 or CCL25 only poorly (Fig. 4C).

CCR4 ligands CCL17 and CCL22 are produced abundantly by CD11chi DCs in the LP
Given the functional expression of CCR4 by LP-Tregs, we next examined the expression of CCR4 ligands (CCL17/CCL22) in the small intestine. Because a previous study reported that mature DCs express CCL17 and CCL22 and attract Tregs in vitro (29), we investigated whether DCs in the intestinal LP (LP-DCs) express these chemokines. As shown in Fig. 5(A), CD11chi LP-DCs expressed abundant mRNAs for CCL17 and CCL22, compared with splenic CD11chi cells. To examine whether CCL17 and CCL22 are expressed at the protein level, we performed ELISAs. As shown in Fig. 5(B), CCL17 and CCL22 were readily detected in the culture medium of LP-DCs and their expression levels were significantly higher than those of splenic DCs and EOs obtained from LP (LP-EOs) under the same culture conditions. Next, we examined the possibility that LP-Tregs could be attracted by LP-DCs. For this purpose, LP-DCs were cultured, and the culture supernatant was harvested and examined for the ability to attract Tregs. We found that culture supernatant obtained from LP-DCs attracted LP-Tregs very efficiently, which was inhibited by the addition of neutralizing antibodies against CCL17 and CCL22 (Fig. 5C), raising the possibility that LP-Tregs can migrate toward LP-DCs via CCR4-mediated signaling.


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. LP-DCs abundantly express CCL17 and CCL22. (A) Expression of CCL17 and CCL22 mRNA in LP-DCs. cDNA was prepared from the total RNA obtained from freshly FACS-sorted LP-derived and spleen-derived DCs, and the expression of CCL17 and CCL22 was analyzed by real-time PCR. (B) Production of CCL17 and CCL22 by LP-DCs, LP-EOs and splenic DCs. The culture medium from these cells was analyzed by ELISA for the production of CCL17 and CCL22. (C) Conditioned medium obtained from the culture of LP-DCs induce chemotaxis in LP CD4+CD25+ Treg cells. LP-DCs were isolated and cultured for 24 h before harvesting the supernatants. Supernatants were tested for their chemotactic activity on CD4+CD25+ Treg cells. White bar shows percentage of migrated LP CD4+CD25+. Isotype-matched control or anti-CCL17 and/or anti-CCL22 mAbs (10 µg ml–1) were added to the supernatants. Results are representative of two independent experiments.

 
Tregs are located in close proximity to DCs in the LP
To examine whether the observed expression of CCL17, CCL22 and CCR4 within the intestinal LP reflected functional interactions between these molecules, we examined the localization of DCs and Tregs in the LP. As shown in Fig. 6, LP-Foxp3+ Tregs (52 ± 5.4%) were often located in close proximity to the CD11c+ LP-DCs, whereas 34 ± 4.2% of Foxp3+ cells were in contact with CD11c+ cells in the spleen (data not shown). These findings are consistent with the hypothesis that Tregs are localized to the small intestinal LP through the action of CCL17 and CCL22 produced locally by LP-DCs.


Figure 6
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. LP-Tregs are found in close proximity to LP-DCs. Frozen sections were stained with antibodies specific for CD11c (red) and Foxp3 (green) and analyzed by confocal microscopy. Foxp3+ Tregs were found adjacent to CD11c+ DCs in the small intestinal LP. Original magnification, x400 (left) and x1200 (right).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Tregs continuously recirculate throughout the secondary lymphoid organs and peripheral tissues to maintain immunological tolerance in vivo. Although it has been generally accepted that Treg migration is under the control of chemokines and chemokine receptors (30), studies of the molecules responsible for the steady-state migration of circulating Tregs to the gut are scarce. In the present study, we found that CD4+CD25+ T cells were abundant in the small intestinal LP and that most of them expressed a critical transcription factor for the development and function of Tregs, Foxp3. Most of the CD4+CD25+ T cells were CD62LloCCR7– and CD44hiCD45RBlo, indicating that they are effector/memory-like Tregs. The LP-Tregs expressed higher levels of CCR9 than did other T-cell subsets in spleen. They also showed a high expression level of CCR4 at the RNA and protein levels compared with splenic and MLN–Tregs and efficiently migrated to the CCR4 ligands CCL17 and CCL22. Interestingly, these chemokines were abundant in the small intestinal LP. Furthermore, the LP-Tregs were closely associated with the CD11c+ LP-DCs in situ, which strongly expressed CCL17 and CCL22. These observations indicate that LP-Tregs are different from the Tregs of other tissues in their expression of a unique phenotype and raise the possibility that the LP-Tregs may localize to the small intestinal LP through the action of chemokines CCL17 and CCL22 produced by LP-DCs.

To the best of our knowledge, our study is the first to demonstrate that the Tregs of the small intestinal LP predominantly display an effector/memory phenotype. In other tissues we examined, such as the MLNs and spleen, Tregs with an effector/memory phenotype represented only a minority, consistent with studies by others (7, 8). The dominance of an effector/memory phenotype in LP-Tregs raises at least two possibilities regarding their recruitment to the intestine. One is that naive-type Tregs migrate into the intestinal LP and are rapidly converted to an effector/memory phenotype locally, in response to the gut-specific environment. Because the small intestine is an antigen-rich milieu, containing food antigens, apoptotic epithelial cells and commensal bacteria, as well as antigen-presenting DCs in the LP (21, 22), the Tregs migrating into the LP are probably exposed to these antigens via DCs and thereby become activated, which may cause them to express this unique phenotype, although non-antigenic tissue-specific factors such as transforming growth factor-β may also contribute to it. A second possibility is that Tregs with an effector/memory phenotype are generated in non-intestinal tissues and migrate preferentially to the intestinal LP. Previous studies by Hamann et al. indicated that CD62L– Tregs could migrate efficiently to non-lymphoid tissue sites, whereas CD62L+ Tregs mainly migrate to secondary lymphoid tissues (7, 8). However, the low expression of β7 integrin and CD62L observed in the LP-Tregs indicates that they would not have a strong tendency to migrate back to the intestine because cells with this phenotype cannot interact with MAdCAM-1, the principal endothelial receptor that regulates lymphocyte trafficking into the intestinal LP (31, 32).

Foxp3+ LP-Tregs were often found in direct contact with or in close proximity to DCs. In addition to providing antigenic stimuli to Tregs, it is possible that LP-DCs provide a non-antigenic signal to them, and the Tregs may also signal the DCs. For instance, given that the LP-DCs produce the CCR4 ligands CCL17/CCL22 and that Tregs located in close by expressing CCR4, the CCL17/CCL22-producing LP-DCs are likely to guide the CCR4-expressing LP-Tregs to their sites of action. The high expression of CCR4 by blood-borne Tregs has been documented previously in humans (29, 33). In addition, because human monocyte-derived DCs cultured in the presence of GM-CSF plus IL-4 produce CCL17 and CCL22, and their production increases in response to inflammatory stimuli (34), the interaction between CCR4 ligands and CCR4 has been suggested to play a role in Treg recruitment to the sites of inflammation (29, 33). In support of this hypothesis, a recent study showed that CCR4-deficient Tregs failed to migrate to the draining MLNs that highly expressed CCR4 ligands CCL17 and CCL22, with the consequence that impaired to suppress pathogenic T cells during early development of colitis in a mouse model of IBD (35). In addition, Sather et al. (36) showed that the Tregs in most non-lymphoid tissues are CCR4+ and mice with CCR4-deficient Tregs develop severe inflammatory disease in the skin and lungs. Our study suggested that the production of CCL17/CCL22 is constitutive in the DCs of the mouse intestinal LP. Thus, CCR4-mediated signaling by DCs is likely to play a role in intestinal Treg trafficking under non-inflammatory conditions as well, although the possibility that CD4+CD25– T cells also utilize the same mechanism is not excluded. This hypothesis is in agreement with a recent suggestion by Matzinger (37) that a tissue (such as the intestine) may be able to educate its resident antigen-presenting DCs such that these DCs, in turn, stimulate certain types of responses from T cells. On the other hand, in light of the recent observation that the acute ablation of Tregs results in the rapid activation and proliferation of DCs (38), a reverse type of Treg–DC interaction is also possible: Tregs may act on DCs to affect their function. Further study is warranted to elucidate the biological significance of the Treg–DC interactions in the intestinal LP.

The regulatory potential of LP CD4+CD25+ T cells was examined in vitro. Although both LP and splenic CD4+CD25+ T cells inhibited T-cell proliferation, LP CD4+CD25+ T cells showed substantially lower suppressive activity than splenic CD4+CD25+ T cells. These data may imply the intrinsically weaker regulatory activities of the LP CD4+CD25+ T cells, although contamination of the LP CD4+CD25+ cell population by activated T cells is not ruled out at present. Future work using CD4+Foxp3+ cells from GFP-Foxp3 mice will help resolve this issue. In addition, the use of the GFP-Foxp3 mice should also address how important the appropriate localization of Tregs is for their suppressive capacity in vivo. A recent study indicates that the immunoregulatory activity of Tregs in ongoing immune reactions requires their trafficking into inflamed tissue (39). It has also been shown that, under conditions of intestinal inflammation, Tregs accumulate and proliferate in the intestinal mucosa (40), in agreement with the idea that Tregs control effector T-cell responses not only in lymph nodes but also in inflamed tissue. Thus, determining whether Tregs exert their suppressive function in the intestinal LP, the MLNs or both is critical.

In conclusion, our data showed that the majority of the Tregs in the intestinal LP was phenotypically unique, expressing an effector/memory phenotype. We also showed that the Tregs functionally express CCR4 and are located in close proximity with CCR4 ligand-producing DCs in the intestinal LP. These observations provide useful information for the possible manipulation of Treg recruitment to the intestinal LP, which may be beneficial for achieving tolerance in autoimmune reactions or for enhancing immune responses against invading pathogens.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Ministry of Education, Culture, Sports, Science and Technology of Japan (18790338, 19041044) to M.H.J.; Advanced Research on Cancer from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17014056) to M. Miyasaka.


   Acknowledgements
 
We thank Reinhold Foerster for the CCR9 mAb, Satoshi Uematsu for technical advice on the real-time PCR and Tamae Kondo and Kousuke Fujimoto for helping us perform immunohistochemistry and cytokine ELISA, respectively. We thank Haruko Hayasaka for helpful comments on the manuscript. We also thank Shinobu Yamashita and Miyuki Komine for their secretarial assistance.

Funding to pay the Open Access publication charges for this article was provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Abbreviations
 
DC, dendritic cell
EO, eosinophil
GM-CSF, granulocyte macrophage colony-stimulating factor
LP, lamina propria
MDC, macrophage-derived chemokine
MLN, mesenteric lymph node
RT, room temperature
TARC, thymus and activation-regulated chemokine
TECK, thymus-expressed chemokine
Treg, regulatory T cell

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: T. Saito Back

Received 4 June 2007, accepted 7 December 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

  1. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. (2004) 22:531.[CrossRef][ISI][Medline]
  2. Wing K, Fehervari Z, Sakaguchi S. Emerging possibilities in the development and function of regulatory T cells. Int. Immunol. (2006) 18:991.[Abstract/Free Full Text]
  3. Wei S, Kryczek I, Zou W. Regulatory T-cell compartmentalization and trafficking. Blood (2006) 108:426.[Abstract/Free Full Text]
  4. Zou L, Barnett B, Safah H, et al. Bone marrow is a reservoir for CD4+CD25+ regulatory T cells that traffic through CXCL12/CXCR4 signals. Cancer Res. (2004) 64:8451.[Abstract/Free Full Text]
  5. Lee I, Wang L, Wells AD, Dorf ME, Ozkaynak E, Hancock WW. Recruitment of Foxp3+ T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor. J. Exp. Med. (2005) 201:1037.[Abstract/Free Full Text]
  6. Curiel TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. (2004) 10:942.[CrossRef][ISI][Medline]
  7. Huehn J, Siegmund K, Lehmann JC, et al. Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. J. Exp. Med. (2004) 199:303.[Abstract/Free Full Text]
  8. Lehmann J, Huehn J, de la Rosa M, et al. Expression of the integrin {alpha}Eβ7 identifies unique subsets of CD25+ as well as CD25– regulatory T cells. Proc. Natl Acad. Sci. USA (2002) 99:13031.[Abstract/Free Full Text]
  9. Makita S, Kanai T, Nemoto Y, et al. Intestinal lamina propria retaining CD4+CD25+ regulatory T cells is a suppressive site of intestinal inflammation. J. Immunol. (2007) 178:4937.[Abstract/Free Full Text]
  10. Makita S, Kanai T, Oshima S, et al. CD4+CD25bright T cells in human intestinal lamina propria as regulatory cells. J. Immunol. (2004) 173:3119.[Abstract/Free Full Text]
  11. Kunkel EJ, Campbell JJ, Haraldsen G, et al. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity. J. Exp. Med. (2000) 192:761.[Abstract/Free Full Text]
  12. Papadakis KA, Prehn J, Nelson V, et al. The role of thymus-expressed chemokine and its receptor CCR9 on lymphocytes in the regional specialization of the mucosal immune system. J. Immunol. (2000) 165:5069.[Abstract/Free Full Text]
  13. Kunkel EJ, Butcher EC. Plasma-cell homing. Nat. Rev. Immunol. (2003) 3:822.[CrossRef][ISI][Medline]
  14. Pabst O, Ohl L, Wendland M, et al. Chemokine receptor CCR9 contributes to the localization of plasma cells to the small intestine. J. Exp. Med. (2004) 199:411.[Abstract/Free Full Text]
  15. Hsieh CS, Zheng Y, Liang Y, Fontenot JD, Rudensky AY. An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat. Immunol. (2006) 7:401.[CrossRef][ISI][Medline]
  16. Nishikawa H, Kato T, Tawara I, et al. Definition of target antigens for naturally occurring CD4+ CD25+ regulatory T cells. J. Exp. Med. (2005) 201:681.[Abstract/Free Full Text]
  17. Watanabe N, Wang YH, Lee HK, Ito T, Cao W, Liu YJ. Hassall's corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus. Nature (2005) 436:1181.[CrossRef][Medline]
  18. Apostolou I, Sarukhan A, Klein L, von Boehmer H. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. (2002) 3:756.[ISI][Medline]
  19. Jordan MS, Boesteanu A, Reed AJ, et al. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. (2001) 2:301.[CrossRef][ISI][Medline]
  20. Seddon B, Mason D. Peripheral autoantigen induces regulatory T cells that prevent autoimmunity. J. Exp. Med. (1999) 189:877.[Abstract/Free Full Text]
  21. Jang MH, Sougawa N, Tanaka T, et al. CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes. J. Immunol. (2006) 176:803.[Abstract/Free Full Text]
  22. Huang FP, Platt N, Wykes M, et al. A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J. Exp. Med. (2000) 191:435.[Abstract/Free Full Text]
  23. Hontsu S, Yoneyama H, Ueha S, et al. Visualization of naturally occurring Foxp3+ regulatory T cells in normal and tumor-bearing mice. Int. Immunopharmacol. (2004) 4:1785.[CrossRef][ISI][Medline]
  24. Hamada H, Hiroi T, Nishiyama Y, et al. Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine. J. Immunol. (2002) 168:57.[Abstract/Free Full Text]
  25. Kishimoto TK, Jutila MA, Berg EL, Butcher EC. Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science (1989) 245:1238.[Abstract/Free Full Text]
  26. Szanya V, Ermann J, Taylor C, Holness C, Fathman CG. The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J. Immunol. (2002) 169:2461.[Abstract/Free Full Text]
  27. Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, Song SY. Retinoic acid imprints gut-homing specificity on T cells. Immunity (2004) 21:527.[CrossRef][ISI][Medline]
  28. Mora JR, Bono MR, Manjunath N, et al. Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells. Nature (2003) 424:88.[CrossRef][Medline]
  29. Iellem A, Mariani M, Lang R, et al. Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4+CD25+ regulatory T cells. J. Exp. Med. (001) 194:847.[CrossRef]
  30. Huehn J, Hamann A. Homing to suppress: address codes for Treg migration. Trends Immunol. (2005) 26:632.[CrossRef][ISI][Medline]
  31. Berg EL, McEvoy LM, Berlin C, Bargatze RF, Butcher EC. L-selectin-mediated lymphocyte rolling on MAdCAM-1. Nature (1993) 366:695.[CrossRef][Medline]
  32. Berlin C, Berg EL, Briskin MJ, et al. {alpha}4β7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell (1993) 74:185.[CrossRef][ISI][Medline]
  33. Iellem A, Colantonio L, D'Ambrosio D. Skin-versus gut-skewed homing receptor expression and intrinsic CCR4 expression on human peripheral blood CD4+CD25+ suppressor T cells. Eur. J. Immunol. (2003) 33:1488.[CrossRef][ISI][Medline]
  34. Penna G, Vulcano M, Roncari A, Facchetti F, Sozzani S, Adorini L. Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells. J. Immunol. (2002) 169:6673.[Abstract/Free Full Text]
  35. Yuan Q, Bromley SK, Means TK, et al. CCR4-dependent regulatory T cell function in inflammatory bowel disease. J. Exp. Med. (2007) 204:1327.[Abstract/Free Full Text]
  36. Sather BD, Treuting P, Perdue N, et al. Altering the distribution of Foxp3(+) regulatory T cells results in tissue-specific inflammatory disease. J. Exp. Med. (2007) 204:1335.[Abstract/Free Full Text]
  37. Matzinger P. Friendly and dangerous signals: is the tissue in control? Nat. Immunol. (2007) 8:11.[CrossRef][ISI][Medline]
  38. Kim JM, Rasmussen JP, Rudensky AY. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. (2007) 8:191.[CrossRef][ISI][Medline]
  39. Siegmund K, Feuerer M, Siewert C, et al. Migration matters: regulatory T-cell compartmentalization determines suppressive activity in vivo. Blood (2005) 106:3097.[Abstract/Free Full Text]
  40. Mottet C, Uhlig HH, Powrie F. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J. Immunol. (2003) 170:3939.[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 arrowOA All Versions of this Article:
20/3/307    most recent
dxm143v1
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 PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Google Scholar
Right arrow Articles by Guo, Z.
Right arrow Articles by Miyasaka, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guo, Z.
Right arrow Articles by Miyasaka, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?