Originally published online as doi:10.1189/jlb.0906574 on August 14, 2007

Published online before print August 14, 2007

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(Journal of Leukocyte Biology. 2007;82:1230-1238.)
© 2007 by Society for Leukocyte Biology

CCR7 mediates the migration of Foxp3⁺ regulatory T cells to the paracortical areas of peripheral lymph nodes through high endothelial venules

Satoshi Ueha^*, Hiroyuki Yoneyama^*, Shigeto Hontsu, Makoto Kurachi^*, Masahiro Kitabatake^*, Jun Abe^*, Osamu Yoshie, Shiro Shibayama, Tetsuya Sugiyama and Kouji Matsushima^*,1

^* Department of Molecular Preventive Medicine and SORST, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;
Second Department of Internal Medicine, Nara Medical University, Nara, Japan;
Department of Microbiology, Kinki University School of Medicine, Osaka, Japan; and
Exploratory Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan

¹ Correspondence: Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: koujim{at}m.u-tokyo.ac.jp

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Thymus-derived forkhead box p3⁺ naturally occurring regulatory T cells (nTreg) are thought to circulate throughout the body to maintain peripheral immunological self-tolerance through interactions with dendritic cells (DCs), resulting in regulation of conventional T cells. However, the chemokine receptors, which are putatively involved in the in vivo migration of nTreg, have not been fully established. Here, we demonstrated that lymph node nTreg preferentially migrated to the paracortical area of lymph nodes after adoptive transfer, where they were observed to make contact frequently with CD8⁺ DCs and CD8^– CD11b^– DCs. This migration of nTreg to the paracortical areas was impaired severely when cells were prepared from CCR7-deficient mice. However, to some extent, CCR7-independent migration of nTreg in such CCR7-deficient mice was also observed, but this occurred mainly in the medullary high endothelial venules. Taken together, these data provide the evidence that CCR7 mediates nTreg migration to the paracortical areas of lymph nodes under steady-state conditions; however, CCR7-independent migration also takes place in the medulla.

Key Words: chemokine • cell migration • medulla • migration • tolerance

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The CD25⁺ CD4⁺ T cell population in normal, naïve animals contains naturally occurring regulatory T cells (nTreg), which are considered to play important roles in maintaining peripheral immunological self-tolerance by suppressing auto-reactive T cells in the periphery [¹ , ² ]. It is generally considered that TCR triggering of nTreg by dendritic cells (DCs) is required for the induction of suppressor function, and cell–cell contact between activated nTreg and CD25^– conventional T cells is indispensable for the suppression of T cell responses in vitro [^{3 4 5} ]. Therefore, appropriate migration of nTreg and subsequent interactions among nTreg, DCs, and conventional T cells are presumed to be essential for the maintenance of immunological self-tolerance in vivo. However, the molecular interactions necessary for nTreg migration and immunoregulatory cellular interactions have not been fully elucidated.

Forkhead/winged helix transcription factor 3 (Foxp3) was described originally as a causative gene of the human X-linked recessive disease immune dysregulation, polyendocrinopathy, and scurfy mice and is now established as a key regulatory gene for the development and function of nTreg [⁶ ]. Moreover, it has become clear that Foxp3 expression correlates with the suppressor activity of CD4⁺ T cells, irrespective of CD25 expression [⁷ ]. Using antibody against mouse Foxp3, we demonstrated previously that Foxp3⁺ nTreg preferentially localized in the paracortical area of lymph nodes, where they made direct contact with Foxp3^– conventional T cells and CD11c⁺ DCs under steady-state conditions [⁸ ].

The migration of immune-competent cells to the lymph nodes does not occur randomly but rather, is a consequence of highly coordinated processes involving the chemokine/chemokine receptor system and adhesion molecules [^{9 10 11} ]. It is well established that interactions between chemokine receptor CCR7 and CCR7 ligands CCL21/secondary lymphoid tissue chemokine (SLC) and CCL19/EBI-1-ligand chemokine are essential for naïve T cells, as well as a fraction of memory T cells, to extravasate across lymph node high endothelial venules (HEV) and migrate to the T cell regions of secondary lymphoid tissues [¹² , ¹³ ]. In addition, CCR7 governs the migration of immature DCs in the tissues to the draining lymph nodes via afferent lymphatics [¹⁴ ]. Although several chemokine receptors, especially CCR7 and CCR4, have been suggested to be important for the migration and suppressor function of nTreg in several autoimmune disease models [^{15 16 17 18 19 20 21 22 23 24} ], the chemokine–chemokine receptor system, which regulates nTreg migration to paracortical areas of lymph nodes under steady-state conditions, has not been fully elucidated.

Here, using CCR7^–/– mice and low molecular weight CCR4 antagonists, we report that the migration of Foxp3⁺ nTreg to the paracortical areas of lymph nodes largely depends on CCR7 but not on CCR4. In addition, a CCR7-independent transmigration of nTreg to lymph nodes was found to occur mainly in the medullary HEV.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mice
C57BL/6 Ly5.2 (CD45.2) and C57BL/6 Ly5.1 (CD45.1) mice were purchased from CLEA Japan Inc. (Shizuoka, Japan) and The Jackson Laboratory (Bar Harbor, ME, USA), respectively. CCR7-deficient C57BL/6 mice [¹² ] were provided by Dr. Martin Lipp (Max Delbrück Center for Molecular Medicine, Germany). All animal procedures described in this study were performed according to the guidelines for animal experiments of the University of Tokyo (Japan).

Flow cytometry and cell sorting
Purified and FITC-, PE-, allophycocyanin-, PerCP-Cy5.5-, PC7-, or biotin-conjugated, anti-mouse mAb to FcR (2.4G2), CD4 (RM4-5), CD25 (PC61 or 7D4), CD45.1 (A20), CD62 ligand (CD62L; MEL-14), CD103 (M290), lymphocyte Peyer’s patch HEV adhesion molecule 1 (DATK32), CCR5 (C34-3448), CXCR4 (2B11), CXCR5 (2G8), and TCR-β (H57-597), as well as subclass-matched, control antibodies and fluorescent dye-conjugated streptavidin, were purchased from BD PharMingen (San Diego, CA, USA). Anti-mouse mAb to CCR6 (140706), CCR9 (242503), CXCR3 (220803), and CXCR6 (221002) were purchased from R&D Systems (Minneapolis, MN, USA). Anti-mouse CCR7 mAb (4B12), F4/80 (BM8), anti-mouse Foxp3 mAb (FJK-16s), and the anti-mouse/rat Foxp3 staining set were purchased from eBioscience (San Diego, CA, USA). Hamster anti-mouse CCR4 mAb (Clone 2G12) [²⁵ ] and anti-mouse CCR8 mAb will be described in detail elsewhere by Nagakubo et al. (unpublished results) Flow cytometry and cell sorting were performed by an EPICS ALTRA cell sorter with EXPO32 software (Beckman Coulter, Fullerton, CA, USA). Dead cells were excluded on the basis of forward- and side-scatter profiles and propidium iodide staining. For the purification of lymph node CD25⁺ CD4⁺ T cells, CD25⁺ cells were enriched by MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and then sorted using the EPICS ALTRA. The purity of sorted cells was routinely >98%.

Immunofluorescence staining
Frozen sections were stained as described previously [⁸ ]. In brief, cryosections were fixed in ice-cold acetone and preincubated in Block Ace (Dainippon Pharmaceutical Co., Ltd, Tokyo, Japan). Subsequently, samples were incubated with primary antibodies or appropriate control antibodies, followed by appropriate Alexa-labeled secondary reagents (Invitrogen Japan K. K., Tokyo, Japan). For the detection of Foxp3, we used rabbit anti-mouse Foxp3 polyclonal antibodies [⁸ ]. The samples were then analyzed using an Olympus IX-70 confocal laser-scanning microscope (Olympus Optical, Tokyo, Japan).

Chemotaxis assay
Chemotaxis assays were performed with the ChemoTx plate (Neuro Probe, Gaithersburg, MD, USA), according to the manufacturer’s instructions. In brief, CD25⁺ CD4⁺ or CD25^– CD4⁺ T cells were suspended at 2 x 10⁶ cells/ml in RPMI 1640 containing 0.5% BSA and 20 mM HEPES. Cell suspensions (25 µl) were loaded onto the membrane plate and placed in a flat-bottomed, 96-well microtiter plate containing 29 µl serially diluted SLC, stromal cell-derived factor 1 (SDF-1)/CXCL12, B lymphocyte chemoattractant (BLC)/CXCL13, thymus and activation-regulated chemokine (TARC)/CCL17, and macrophage-derived chemokine (MDC)/CCL22 in triplicate.

Adoptive transfer
For the kinetic study of the migration of Foxp3⁺ nTreg to peripheral lymphoid tissue, 3 x 10⁷ of CD45.1⁺ lymph node cells were transferred to CD45.2 recipient mice via tail vein, and the CD45.1⁺ Foxp3⁺ CD4⁺ T cells in the peripheral blood, peripheral lymph nodes (axillary, inguinal, submandibular), mesenteric lymph node, and the spleen of recipient mice were analyzed at 0.5, 2, 6, 24, and 48 h after transfer. In some experiments, donor cells were preincubated with anti-CD62L mAb (10 µg/ml) or control antibodies for 10 min at 37ºC before transfer. To test the role of CCR7 in nTreg migration, an equal number (1x10⁷ cells) of CCR7^+/+ CD45.1⁺ or CCR7^–/– CD45.2⁺ spleen CD4 T cells was injected i.v. into (Ly5.1xLy5.2) F1 recipient mice. In some experiments, recipient mice were s.c.-injected with 10 mg/kg AMD3100 20 min before transfer [²⁶ ]. Two hours later, mice were perfused with 50 ml PBS, and mononuclear cells collected from the peripheral blood, peripheral lymph nodes, mesenteric lymph node, and spleen were analyzed by flow cytometry. For the immunohistological analysis, 3–8 x 10⁶ spleen CD25⁺ CD4⁺ T cells from CCR7^+/+ or CCR7^–/– mice were labeled with CFSE (20 µM) and then transferred to wild-type recipient mice, which were killed and perfused with 50 ml PBS at 2 h or 24 h after transfer. The axillary, inguinal, and submandibular lymph nodes were collected, and their 12-µm cryosections were subjected to immunohistological analysis. We regarded the TCR-β⁺ T cell-rich region as paracortex, B220⁺ B cell-rich region as cortex, and F4/80⁺ macrophage-rich region as medulla. To test the role of CCR4 in nTreg migration, CD45.2⁺ mice were administered orally with CCR4 antagonist (30 mg/Kg) or control solvent (0.5% methylcellulose solution) and then i.v.-injected with CD45.1⁺ spleen cells. Two hours after transfer, the migration of CD45.1⁺ Foxp3⁺ nTreg was analyzed by flow cytometry.

CCR4 antagonist
The CCR4 antagonist was generated as described previously [²⁷ ]. This reagent specifically inhibits chemotactic responses of mouse CCR4-transduced L1.2 cells to mouse TARC (10^–8 M) at a concentration of 10^–8 M, and oral administration of the CCR4 antagonist at a dose of 30 mg/Kg completely inhibits in vivo cell recruitment of Th2 cells toward MDC for at least 12 h in a mouse air-pouch model [²⁸ ].

Statistical analysis
Data are presented as means with SD. Statistical comparisons between groups were made using the Student’s t-test. P < 0.05 was considered to be statistically significant.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Migration properties and kinetics of Foxp3⁺ nTreg to peripheral lymphoid tissues
We demonstrated previously that Foxp3⁺ nTreg are localized in the paracortical area of lymph nodes in direct contact with Foxp3^– conventional T cells and DCs [⁸ ]. To gain further understanding about the molecular requirement for the migration of Foxp3⁺ nTreg to the paracortical area of peripheral lymph nodes, we first performed adoptive transfer experiments using the CD45.1/CD45.2 congenic system and analyzed the migration properties and kinetics of Foxp3⁺ nTreg (Fig. 1A ). It has been reported that the migration efficiency of CD25⁺ CD4⁺ T cells to lymphoid tissues is relatively low in comparison with conventional T cells [²⁹ ]. Consistent with the previous report, the recovery of transferred CD45.1⁺ Foxp3⁺ CD4⁺ nTreg was 20% of CD45.1⁺ Foxp3^– CD4⁺ conventional T cells in the peripheral lymph nodes, mesenteric lymph node, and spleen at 2 h after adoptive transfer (Fig. 1B and 1C) . The poor recovery of Foxp3⁺ nTreg was observed at 0.5 h, and it persisted for an observation period of 48 h after transfer (data not shown). Kinetic study revealed that the number of CD45.1⁺ Foxp3⁺ nTreg in the peripheral blood was equilibrated within 2 h after transfer, suggesting that the distribution of transferred Foxp3⁺ nTreg from the peripheral blood to tissue occurred within 2 h. In the peripheral and mesenteric lymph nodes, the number of CD45.1⁺ Foxp3⁺ nTreg reached the peak at 24 h, whereas in the spleen, it occurred at 6 h and was followed by a slight decrease (Fig. 1D) . The kinetics suggests that the spleen migrating Foxp3⁺ nTreg, which might in part be trapped via an open vessel system, recirculated and redistributed to peripheral lymphoid tissues thereafter. It is important that pretreatment of Foxp3⁺ nTreg with anti-CD62L-blocking antibodies significantly inhibited the migration of Foxp3⁺ nTreg to the peripheral lymph nodes at 2 h (Fig. 1E 1F 1G) , suggesting that this migration is L-selectin-mediated transendothelial migration. Therefore, we mainly analyzed the migration of Foxp3⁺ nTreg at 2 h in most of the adoptive transfer experiments to focus on the direct transendothelial migration of Foxp3⁺ nTreg in the peripheral lymph nodes.

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Figure 1. Migration properties of Foxp3+ nTreg to secondary lymphoid tissues. (A) CD45.1+ lymph node cells were adoptively transferred to CD45.2 recipient mice. At various time-points, the mononuclear cells from the peripheral blood (PB), peripheral lymph nodes (PLN; axillary, inguinal, submandibular), mesenteric lymph nodes (MLN), and spleen were stained with intracellular Foxp3, CD4, and CD45.1 and assessed by flow cytometry. (B) Representative flow cytometry profiles of lymph node cells at 2 h after transfer. The values in each histogram represent the mean percentage of CD45.1+ donor cells (n=3). (C) Percent recovery of Foxp3+ nTreg and Foxp3– conventional CD4 T cells to the transferred cells in each tissue at 2 h after transfer (n=3; **, P< 0.01). (D) Kinetics of the number of CD45.1+ Foxp3+ CD4+ T cells in each tissue (n=3 for each time-point). Representative data from two independent experiments are shown. (E) CD45.1+ lymph node cells were preincubated with anti-CD62L mAb or control rat IgG2a and then transferred to CD45.2 recipient mice. (F) Representative flow cytometry profiles of CD45.1 expression in the lymph node Foxp3+ CD4+ cells at 2 h after adoptive transfer. The values in each histogram represent the mean percentage of CD45.1+ donor cells (n=3). (G) The number of CD45.1+ Foxp3+ CD4+ T cells in the peripheral lymph node and spleen (SPL) of the recipient mice of anti-CD62L mAb or rat IgG2a-treated lymph node cells (n=3 for each group; *, P=0.012; **, P<0.01). Representative data from two independent experiments are shown.

Distribution of Foxp3⁺ nTreg in the peripheral lymph nodes
To quantify the distribution of Foxp3⁺ nTreg in the peripheral lymph nodes, CD25⁺ CD4⁺ T cells were purified from lymph nodes and labeled with CFSE and then adoptively transferred to recipient mice via tail vein. Two hours after adoptive transfer, the distribution of CFSE⁺ transferred, and CFSE^– intrinsic Foxp3⁺ nTreg were analyzed by confocal laser microscopy. As shown in Figure 2A and 2B , transferred and intrinsic Foxp3⁺ nTreg were found mainly in the paracortex of the lymph nodes, as well as some in the medulla and cortex (paracortex: transferred 92%, intrinsic 91%; medulla: transferred 5.8%, intrinsic 6.3%; cortex: transferred 2.1%, intrinsic 2.5%). Transmigration of Foxp3⁺ nTreg across HEV was detected from 0.5 h after adoptive transfer (Fig. 2A) . It has been reported that nTreg stably interact ex vivo with CD8^– CD11b^lo/– DC subsets in an antigen-dependent manner [³⁰ ]. To determine the DC subsets with which nTreg preferentially clustered in the peripheral lymph nodes in vivo, we next analyzed contacts between Foxp3⁺ nTreg and DC subsets. As the number of clusters between transferred Foxp3⁺ nTreg and CD11c⁺ DCs was difficult to quantify precisely, even at 24 h, and the distribution of transferred Foxp3⁺ nTreg was essentially similar to that of intrinsic Foxp3⁺ nTreg, we analyzed the intrinsic Foxp3⁺ nTreg–DC clusters in the peripheral lymph nodes. Immunohistological analysis revealed that in the paracortical area, Foxp3⁺ nTreg were frequently found to be in contact with CD8^– CD11b^– CD11c⁺ DCs and CD8⁺ CD11c⁺ DCs but less frequently with CD11b⁺ CD11c⁺ DCs (46%, 35%, 23%, respectively, Fig. 2C and 2D ).

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Figure 2. Distribution and cellular interactions of Foxp3+ nTreg in the peripheral lymph nodes, and lymph node CD25+ CD4+ T cells were labeled with CFSE and i.v.-injected into untreated recipient mice. Two hours after transfer, the cryosections of axillary lymph nodes were prepared and analyzed by confocal microscopy. (A) Distribution of CFSE+ Foxp3+ nTreg 2 h after adoptive transfer (green, CFSE; red, Foxp3; blue, type IV collagen). M, Medulla; P, paracortex; C, cortex. Yellow circles indicate the CFSE+ Foxp3+ cells. Small window shows the transendothelial migration of CFSE+ Foxp3+ nTreg at 0.5 h after transfer [green, CFSE; red, Foxp3; blue, peripheral node addressin (PNAd)]. Yellow arrow indicates CFSE+ Foxp3+ nTreg. (B) The distribution of CFSE+ transferred or CFSE– intrinsic Foxp3+ nTreg. Ten nonconsecutive sections were analyzed, and at least 200 CFSC+ Foxp3+cells were counted for each recipient mouse. Representative data from two independent experiments are shown. (C) Interaction between intrinsic Foxp3+ nTreg and DC subsets in the paracortical areas of the peripheral lymph nodes (upper: green, Foxp3; red, CD8; blue, CD11c; lower: green, Foxp3; red, CD8/CD11b; blue, CD11c). (D) Percentage of Foxp3+ nTreg–DC subset clusters in total Foxp3+ nTreg–CD11c+ DC clusters in the paracortical area of the peripheral lymph nodes. Approximately 200 Foxp3+ nTreg–CD11c+ DC clusters were evaluated on 20 nonconsecutive sections.

Distribution of Foxp3⁺ nTreg in nonlymphoid tissues
As limited information is available about the distribution of Foxp3⁺ nTreg in nonlymphoid tissues, we next addressed whether they clustered with DCs and conventional T cells in peripheral nonlymphoid tissues under a steady-state condition. In the liver, lung, kidney, and large intestine, we detected a few Foxp3⁺ nTreg but could hardly detect the direct contact between Foxp3⁺ nTreg and Foxp3^– conventional T cells over 10 nonconsecutive sections. In contrast, Foxp3⁺ nTreg were detected frequently in the lamina propria of small intestine, and some of these cells were in direct contact with Foxp3^– conventional T cells and CD11c⁺ DCs (Fig. 3A and 3B ).

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Figure 3. Distribution of Foxp3+ nTreg in peripheral nonlymphoid tissues. (A) Cryosections of liver, lung, kidney, large intestine (Colon), and small intestine (Jejunum) from 8-week-old, female C57BL/6 mice were stained with antibodies against Foxp3 (green), CD4 (red), and type IV collagen (blue) and analyzed by confocal microscopy. (B) High magnification image of small intestine stained with Foxp3 (green), CD4 (red), and CD11c (blue). PV, Portal vein; TB, terminal bronchiole; M, medulla; L, lumen. Representative results of at least 10 nonconsecutive sections from three mice are shown.

Chemokine receptor expression and chemotactic responses of Foxp3⁺ nTreg
Previous studies suggested that the chemokine receptors CCR7 and CCR4 play an important role in the migration and effective suppressor function of nTreg in vivo [^{18 19 20 21 22 23 24} ]. To determine the role of chemokine receptors in the migration of Foxp3⁺ nTreg to the peripheral lymph nodes, we first analyzed the expression of chemokine receptors on the lymph node-derived Foxp3⁺ nTreg by flow cytometry. As shown in Figure 4A , 80% of Foxp3⁺ nTreg expressed CCR7 at a high level. In addition to CCR7, a part of Foxp3⁺ nTreg also expressed several chemokine receptors including CCR4, CCR8, CXCR4, and CXCR5 at low levels as described previously [¹⁶ ]. In an in vitro chemotactic assay, purified CD25⁺ CD4⁺ T cells (95% of which were Foxp3⁺) showed strong chemotactic responses to CCL21/SLC and significant chemotactic responses to CXCL12/SDF-1 and CXCL13/BLC but weak responses to the CCR4 ligands CCL17/TARC and CCL22/MDC (Fig. 4B and 4C) .

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Figure 4. Chemokine receptor expression and chemotactic response of Foxp3+ nTreg. (A) Mononuclear cells from peripheral lymph nodes were stained with antibodies against intracellular Foxp3, CD4, and chemokine receptors and analyzed by flow cytometry. Shaded histograms indicate background staining with isotype control, dashed lines indicate the Foxp3– CD4+ T cells, and solid lines indicate the Foxp3+ CD4+ T cells. The numbers in each histogram represent the percentage of positive cells in Foxp3+ (upper) and Foxp3– cells (lower), respectively. (B) Cytospin slides of highly purified CD25+ CD4+ T cells were stained with Foxp3 (green) and CD4 (red). (C) Chemotaxis responses of CD25+ CD4+ T cells to SLC, SDF-1, BLC, TARC, and MDC were evaluated using a Boyden chamber. The graph shows the mean of triplicate measurements of the percentage of input cells migrated. Representative data from two independent experiments are shown.

A dominant role for CCR7 in the migration of Foxp3⁺ nTreg to lymph nodes
We next performed the adoptive transfer experiments to examine the role of CCR7 and CCR4 in the migration of Foxp3⁺ nTreg to the lymph nodes. A mixture of equal numbers of CCR7^+/+ CD45.1⁺ and CCR7^–/– CD45.2⁺ spleen CD4 T cells was i.v.-injected into (CD45.1xCD45.2) F1 recipient mice, and the migration of donor Foxp3⁺ nTreg was analyzed at 2 h after transfer (Fig. 5A ). The origin of Foxp3⁺ nTreg could be identified as recipient (CD45.1⁺ CD45.2⁺), CCR7^+/+ donor-derived (CD45.1⁺ CD45.2^–), or CCR7^–/– donor-derived (CD45.1^– CD45.2⁺). As shown in Figure 5B 5C 5D , the recovery of CCR7^–/– Foxp3⁺ nTreg in the peripheral and mesenteric lymph nodes was 10.8 ± 0.2 and 11.1 ± 0.2% of CCR7^+/+ Foxp3⁺ nTreg, respectively. Conversely, there was a concomitant increase in the ratio of CCR7^–/– Foxp3⁺ nTreg in the spleen and peripheral blood. Foxp3^– conventional CD4⁺ T cells from CCR7^–/– mice also showed decreased migration to the peripheral and mesenteric lymph nodes (data not shown), as reported previously [¹² ]. These results suggest that CCR7 plays a pivotal role in the migration of Foxp3⁺ nTreg as well as conventional T cells to the lymph nodes. It is important that the expression of chemokine receptors on CCR7^–/– spleen Foxp3⁺ nTreg was not different from that of CCR7^+/+ spleen Foxp3⁺ nTreg. Although the CD62L expression was down-regulated in CCR7^–/– Foxp3⁺ nTreg, which might also contribute to the reduced migration efficiency of CCR7^–/– Foxp3⁺ nTreg to the peripheral lymph nodes, the 40% reduction of the CD62L^hi population cannot account for the 90% reduction of the migration efficiency of CCR7^–/– Foxp3⁺ nTreg to the peripheral lymph nodes (Fig. 5E) . We also examined the involvement of CCR4 in the migration of Foxp3⁺ nTreg to the peripheral lymph nodes. CD45.1⁺ spleen cells were adoptively transferred to the control- or CCR4 antagonist-treated, CD45.2 recipient mice, and the migration of CD45.1⁺ Foxp3⁺ nTreg was analyzed by flow cytometry 2 h after transfer (Fig. 5F) . As shown in Figure 5G , CCR4 antagonist treatment did not affect the number of CD45.1⁺ Foxp3⁺ nTreg in the peripheral and mesenteric lymph nodes, spleen, and peripheral blood. Thus, the migration of Foxp3⁺ nTreg to the lymph nodes is independent of CCR4 under steady-state conditions.

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Figure 5. CCR7 but not CCR4 mediates the migration of nTreg to the lymph nodes. (A) An equal mixture of CCR7+/+ CD45.1+ and CCR7–/– CD45.2+ spleen CD4 T cells was injected i.v. into (CD45.1xCD45.2) F1 recipient mice. (B) Two hours later, mononuclear cells from the peripheral blood, spleen, peripheral lymph nodes, and mesenteric lymph nodes were stained with antibodies against intracellular Foxp3, CD4, CD45.1, and CD45.2 and analyzed by flow cytometry. Numbers represent the percentage of gated cells. (C) Absolute numbers of CCR7+/+ or CCR7–/– transferred CD4+ Foxp3+ cells recovered from each compartment (n=3; **, P<0.01). (D) The CCR7–/–:CCR7+/+ ratios in transferred CD4+ Foxp3+ cells in each compartment. Representative data from two independent experiments are shown. (E) Expression of chemokine receptors and adhesion molecules on CCR7+/+ and CCR7–/– spleen Foxp3+ CD4+ nTreg. Shaded histograms indicate background staining with isotype control, dashed lines indicate the CCR7+/+, and solid lines indicate CCR7–/– Foxp3+ CD4+ T cells. The numbers in each histogram represent the percentage of positive cells in CCR7+/+ (upper) and CCR7–/– Foxp3+ CD4+ T cells (lower), respectively. Representative data from two independent experiments are shown. (F) CD45.2+ mice were given a CCR4 antagonist or control solvent [0.5% methylcellulose solution (MC)] orally and i.v.-injected with CD45.1+ spleen cells. Two hours after transfer, the absolute numbers of CD45.1+ CD4+ Foxp3+ cells recovered from each compartment were analyzed by flow cytometry (n=3 for each group). Representative data from two independent experiments are shown.

CCR7 mediates the migration of Foxp3⁺ nTreg to the paracortical areas of the lymph nodes
Although CCR7 plays a dominant role in the migration of Foxp3⁺ nTreg to the lymph nodes, some CCR7^–/– Foxp3⁺ nTreg could still migrate into them. It has been suggested that the interactions of the adhesion molecules, which are essential for the transmigration of T cells to the peripheral lymph nodes, are spatially different in the paracortex and medulla [³¹ ]. Therefore, we next examined whether paracortex and medulla have different requirements for CCR7 in Foxp3⁺ nTreg transmigration. CCR7^–/– CD25⁺ CD4⁺ T cells were labeled with CFSE and i.v.-injected to wild-type mice. The distribution of CFSE⁺ Foxp3⁺ nTreg in the peripheral lymph nodes was analyzed at 2 h or 24 h after adoptive transfer. Although the CCR7^+/+ Foxp3⁺ nTreg were predominantly found in the paracortical areas of peripheral lymph nodes (Fig. 2A and 2B) , CCR7^–/– Foxp3⁺ nTreg were mainly distributed in the medulla at 2 h and 24 h after adoptive transfer (Fig. 6A 6B 6C 6D 6E ). CCR7^–/– Foxp3⁺ nTreg were already detected in the medullary cord or adjacent to the medullary HEV but not in the medullary sinus at 2 h after adoptive transfer, and this migration was inhibited by anti-CD62L pretreatment (Fig. 6D and 6F) . These results suggest that the distribution of CCR7^–/– Foxp3⁺ nTreg in the medulla represents the direct extravasation of these cells through medullary HEV but not the translocation of extravasating cells in the paracortical area or the entry through the afferent lymphatics.

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Figure 6. CCR7-independent transmigration of nTreg in the medulla and cortex of the peripheral lymph nodes. CCR7–/– spleen CD4+ CD25+ T cells were labeled with CFSE and i.v.-injected into recipient mice. (A) Twenty-four hours (A–C) or 2 h (D) after transfer, axillary, inguinal, and submandibular lymph nodes of recipient mice were subjected to immunofluorescent staining and analyzed by confocal microscopy. (A) Green, CFSE; red, Foxp3; blue, type IV collagen. (B) Green, CFSE; red, TCR-β; blue, type IV collagen. (C) Green, CFSE; red, F4/80; blue, type IV collagen. Yellow circles indicate CFSE+ cells. White line indicates the medullary area. M, medulla; P, paracortex; C, cortex; MC, medullary cord; MS, medullary sinus. (A) x100 original; (B, C) x200 original; (D) x1200 original. (E) The distribution of transferred CCR7+/+ or CCR7–/– CFSE+ Foxp3+ nTreg in the peripheral lymph nodes was quantified at 24 h after adoptive transfer. Pooled data of three independent experiments. At least 50 cells were counted for each recipient mice. (F) CCR7–/– CD45.2+ lymph node cells were preincubated with anti-CD62L mAb or control rat IgG2a and then transferred to CD45.1 recipient mice. Two hours after transfer, the number of CD45.2+ Foxp3+ CD4+ cells was analyzed by flow cytometry (n=3 for each group; *, P=0.020; **, P<0.01). (G) CD45.1 mice were s.c.-injected with CXCR4 antagonist AMD3100 or control PBS and then i.v.-injected with CCR7–/– CD45.2+ spleen cells. Two hours after adoptive transfer, the absolute numbers of CD45.2+ CD4+ Foxp3+ cells in the peripheral lymph nodes and the spleen were analyzed by flow cytometry (n=3 for each group; *, P=0.047; **, P<0.01). Representative data from two independent experiments are shown.

CXCR4 mediates CCR7-independent migration of Foxp3⁺ nTreg to peripheral lymph nodes
CCR7 was required for the transmigration of Foxp3⁺ nTreg across HEV in the paracortical area but not for the transmigration through the medullary HEV. Therefore, we addressed which chemokine receptors are involved in the CCR7-independent migration of Foxp3⁺ nTreg to the peripheral lymph nodes. It has been reported that in plt/plt mice, which lack the genes for CCL19 and CCL21-ser, migration of CXCR4-deficient T cells to peripheral lymphoid tissues was severely impaired [³² ]. Therefore, we next examined whether the CXCR4 antagonist AMD3100 would reduce the migration of CCR7^–/– nTreg to peripheral lymph nodes. A mixture of equal number of CCR7^+/+ CD45.1⁺ and CCR7^–/– CD45.2⁺ spleen CD4 T cells was i.v.-injected into the control (PBS)-treated or AMD3100-treated (CD45.1xCD45.2) F1 recipient mice, and the migration of donor Foxp3⁺ nTreg was analyzed at 2 h after transfer. As expected, AMD3100 treatment significantly reduced the accumulation of CCR7^–/– Foxp3⁺ nTreg in the peripheral lymph nodes to 40 ± 13% of the control-treated recipients. In contrast to peripheral lymph nodes, AMD3100 treatment increased the accumulation of CCR7^–/– Foxp3⁺ nTreg in the spleen (Fig. 6G) . These data suggest that the transmigration of CCR7^–/– Foxp3⁺ nTreg in the peripheral lymph nodes is partially dependent on CXCR4.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although the molecular mechanisms involved in the suppressor function of nTreg are a topic of great interest, relatively little attention has been paid to their in vivo migration and localization. Here, we have analyzed the chemokine receptor use of nTreg for the entry into the peripheral lymph nodes via HEV. Our results show that CCR7 is indispensable for the transmigration of nTreg through HEV into the paracortical areas of the lymph nodes, whereas the transmigration of nTreg through medullary HEV can occur in the absence of CCR7. The migration of CCR7^–/– nTreg to peripheral lymph nodes was inhibited by treatment with a CXCR4 antagonist, suggesting that CXCR4 partly mediates the CCR7-independent migration of nTreg to peripheral lymph nodes.

Under steady-state conditions, direct contact among nTreg, conventional T cells, and DCs cells mainly occurred in the paracortical areas of the secondary lymphoid tissues. It has been reported that nTreg expressing L-selectin, a critical lymphoid homing receptor, efficiently protect mice from lethal acute graft-versus-host disease and delay diabetes in prediabetic NOD mice, whereas L-selectin-negative nTreg do not have these effects [¹⁸ , ¹⁹ ]. In addition, Tang et al. [³⁰ ] demonstrated recently that self-antigen-specific nTreg are constantly being activated by tissue-derived DCs, preventing the priming of autoreactive, pathogenic cells in the antigen-draining lymph node under steady-state conditions. These reports indicate that the specific migration of nTreg to lymph nodes is important for the effective suppression of immune responses in vivo.

Among the DC subsets, nTreg have been reported to stably interact with tissue-derived, antigen-bearing DCs negative for or expressing only low levels of CD11b without CD8 expression [³⁰ ]. In our flow cytometry analysis, CCR7-dependent, tissue-derived DCs (CD11c⁺ MHC II^hi) in the skin-draining lymph nodes [¹⁴ ] were negative for CD8 and low-to-negative for CD11b (data not shown). This is important, as these DCs are suggested to be required for the maintenance of peripheral tolerance through the induction of T cell tolerance. Thus, interactions between nTreg and CD8^– CD11b^– CD11c⁺ DCs in the paracortical areas of the lymph nodes may represent an interaction between nTreg and tissue-derived DCs. In addition, CD8⁺ DCs, which are reported to be important for peripheral cross-tolerance to soluble antigen [³³ ], also clustered with nTreg in the paracortical area. The significance of the interaction between nTreg and CD8⁺ DCs for maintenance of peripheral tolerance remains to be determined.

Our data suggested that CCR7 plays an essential role in the transmigration of nTreg in the paracortical area but not in the medulla of peripheral lymph nodes through HEV. Schneider et al. [²⁴ ] demonstrated recently that CCR7 expression by nTreg was required for the migration of nTreg to peripheral lymph nodes and contributes to their suppressive function in vivo. In addition to nTreg, CCR7 mediates the migration of conventional T cells and tissue-derived DCs to the paracortical area of peripheral lymph nodes [¹² , ¹⁴ ]. In these paracortical areas, CCL21 is produced abundantly by stromal cells in addition to endothelial cells of HEV. Therefore, CCR7/CCL21 interactions may guide nTreg, conventional T cells, and antigen-bearing, tissue-derived DCs into the areas rich in CCL21-producing stromal cells and may constitute an immunoregulatory niche. It is important that CCR7^–/– mice exhibit an autoimmune-like phenotype including autoimmune gastritis and colitis, and the abnormal peripheral circulation of lymphocytes or the abnormal thymic differentiation of CCR7^–/– T cells is suggested to be responsible for the autoimmune-like phenotype in these mice [³⁴ , ³⁵ ]. Further studies are required to elucidate the role of CCR7 in the maintenance of peripheral, immunological tolerance.

Although CCR7/CCR7 ligand interactions are essential for primary T cell immunity [¹² ], CCR7^–/– mice and plt mice have been reported to exhibit delayed but eventually comparable or even enhanced T cell immune responses [³⁶ , ³⁷ ]. Therefore, CCR7-independent medullary migration of nTreg may play a role in the regulation of T cell responses under inflammatory conditions in which CCR7-negative effector type T cells are abundantly induced, although this migration may not play a role under steady-state conditions. The cellular components, cellular interactions, antigen delivery, and biological significance of medullary reactions must be investigated further.

 
This work was supported in part by Solution Oriented Research for Science and Technology (SORST) and the Japan Science and Technology Corporation (JST) and a grant from the Japan Society for the Promotion of Science. We thank Dr. Martin Lipp (Max Delbrück Center for Molecular Medicine) for providing CCR7-decifient mice. We are grateful to Drs. T. Shimaoka, S. Hashimoto, K. Kakimi, and S. Ishikawa for scientific discussions and to S. Fujita, A. Nakano, E. Toda, S. Takao, and S. Shawkat for their kind assistance.

Received September 18, 2006; revised July 2, 2007; accepted July 4, 2007.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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