Pivotal role of CCL25 (TECK)CCR9 in the formation of gut cryptopatches and consequent appearance of intestinal intraepithelial T lymphocytes
Nobuyuki Onai1,
Masahiro Kitabatake2,
Yan-yun Zhang1,
Hiromichi Ishikawa3,
Sho Ishikawa1 and
Kouji Matsushima1
1 Department of Molecular Preventive Medicine and Core Research for Evolutional Science and Technology (CREST), Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-033, Japan 2 Department of Hygiene Chemistry, Faculty of Pharmaceutical Science, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-0826, Japan 3 Department of Microbiology, Keio University, School of Medicine, 35 Shinanomachi, Shinjyuku-ku, Tokyo 160-8582, Japan
Correspondence to: K. Matsushima; E-mail: koujim{at}m.u-tokyo.ac.jp
Transmitting editor: S. Koyasu
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Abstract
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Cryptopatches (CP) are murine gut anatomical sites for generating thymus-independent intraepithelial T lymphocytes (IEL). However, it remains elusive how lympho-hematopoietic progenitor cells migrate from bone marrow (BM) into CP and differentiate into IEL. Here we show that mice reconstituted with BM-derived c-kit+ cells express CCL25 (TECK)-intrakine gene, which reduces specifically the chemotactic response to CCL25 but not CXCL12 in the thymocytes. These mice exhibited a dramatic reduction of CP and IEL in the small intestine, and harbored conspicuously decreased numbers of c-kit+ cells in the emaciated CP. In contrast, T cells in the thymic, splenic and lymph node compartments developed normally in these mice. Importantly, it was demonstrated that CD11c+ dendritic stromal cells in CP expressed CCL25 and c-kit+ Lin BM cells displayed vigorous chemotactic response to CCL25. Furthermore, RT-PCR analysis detects mRNA expression of CCR9 in the c-kit+ Lin BM cells. Thus, these results demonstrate that the CCL25CCR9 pathway is essential for CP formation and the consequent appearance of IEL.
Keywords: CCL25, CCR9, cryptopatches, intracellular chemokine, intraepithelial T lymphocytes
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Introduction
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Gut-associated lymphoid tissue (GALT) in the mouse small intestine consists of Peyers patches (PP), isolated lymphoid follicles, lamina propria lymphocytes, intraepithelial lymphocytes (IEL) (1) as well as recently identified cryptopatches (CP) (2). GALT is continuously exposed to a variety of luminal antigens and comprises one of the largest compartments of the immune system (3). Numerous IEL bearing either TCR
ß or TCR
are localized in the anatomical front between columnar epithelial cells of the small intestine. They form a population markedly different from T cells that develop in the thymus and are distributed in peripheral lymphoid tissues (46).
In contrast to thymus-derived T cells, for instance, most 
-IEL and many
ß-IEL express the unique CD8
homodimmer and develop without passing through the thymus. CP were identified as multiple tiny clusters filled with c-kit+ IL-7R+ Thy-1+ lympho-hematopoietic progenitors in the crypt of the murine small intestine and have been shown to be intestinal lymphoid tissues where clusters of precursor IEL develop (7,8).
Cytokine common receptor (CR)
chain-deficient mice exhibit lymphoid abnormality, severely impaired IEL development and lack the CP (9). On the other hand, CP is not altered in athymic nude, SCID, TCRß x TCR
/, RAG-2/ and aly/aly mutant mice, and is comparable with that in normal mice (2). However, it remains an open question how lympho-hematopoietic progenitors reach the crypt of the lamina propria, rearrange their TCR genes and differentiate into mature IEL.
The CC chemokine CCL25 (TECK) was originally cloned from a thymus cDNA library derived from RAG-1-deficient mice using the random sequencing method (10), and has been shown to be expressed in stromal cells in the thymus and crypt epithelium in the small intestine, but not in the colonic epithelium (11). CCR9, the receptor for CCL25, is expressed on various subsets of thymocytes (1216), and intestinal homing CD4+ and CD8+ T cells (17). These results suggest that the CCL25CCR9 system might be important for the migration of T cells within the thymus during clonal selection and specialized localization of T cells engaged in intestinal mucosal immunity. However, the majority of these results so far have been obtained by in vitro studies and the physiological significance of the CCL25CCR9 pathway in vivo remains poorly understood.
The intrakine (IK) is a genetically modified intracellular chemokine that is linked with an endoplasmic reticulum retention signal sequence (KDEL). It has been shown that IK binds the cognate chemokine receptor(s) at intracellular sites and prevents the cell surface expression of the receptor(s) in vivo as well as in vitro (1820).
The reconstituted mice with the SDF-1-IK gene lacked responsiveness against SDF-1 and exhibited severely impaired hematopoiesis including T lymphopoiesis (20). IK may have an advantage for therapeutic use for hematopoietic disease including AIDS, as well as for functional analysis of particular cytokines in vivo in adult stage.
Here we provide evidence that the CCL25CCR9 system has a critical role in CP generation and IEL development. We first show the significant reduction of the number of CP and IEL in mice reconstituted with bone marrow (BM)-derived hematopoietic progenitor cells that were transduced with a genetically modified CCL25-IK-expressing gene, and then demonstrate that CCL25 is produced by stromal dendritic cells in the CP and is a chemoattractant for c-kit+ Lin cells in BM.
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Methods
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Construction of a bicistronic expression vector and generation of reconstituted mice
Construction of a bicistronic retroviral expression vector containing green fluorescent protein (GFP) and internal ribosomal entry site (IRES) genes has been described (20). Total RNA from thymus of C57BL/6 mouse was used for isolation of murine CCL25 cDNA. The murine CCL25 gene was amplified by RT-PCR reaction with the primers 5'-GCG GATCCACCATGAAACTGTGCCTTTTTG-3' and 5'-GCGAAT CCTTAATTGTTGGTCTTTCTGGGC-3'. The murine CCL25 gene was linked with an endoplasmic reticulum retention signal (SEKDEL) by a PCR reaction with the primers 5'-GCGGATCCACCATGAAACTGTGCCTTTTTG-3' and 5'-GCGAATTCTTACAGCTCGTCCTTCTCGCTATTGTTGGTCTTTCTGGGC-3'. These DNA fragments were digested with BamHI and EcoRI, and inserted into the BamHIEcoRI site of expression vector. The production of supernatants containing retroviruses, retrovirus-mediated BM transduction and generation of reconstituted mice was described previously (20). Briefly, c-kit+ cells from BM were precultured with serum-free medium SF03 (Sankou-junyaku, Tokyo, Japan) containing 10 ng/ml murine stem cell factor (SCF), 10 ng/ml murine IL-6 and 10 ng/ml human Flt3L for 48 h. Cells (1 x 106) cells were transduced by centrifugation with 2 ml retrovirus supernatants containing 10 ng/ml murine SCF, 10 ng/ml mIL-6, 10 ng/ml hFlt3L and 8 µg/ml polybrene (Sigma, St Louis, MO) at 2500 g for 2 h at 2830°C on day 1, and then cultured with DMEM containing 10% FCS, 10 ng/ml murine SCF, 10 ng/ml mIL-6 and 10 ng/ml hFlt3L for 22 h.
On day 2, cells were also transduced by the centrifugation method with retrovirus supernatants. On day 3, GFP+ cells were purified using an Epics Elite ESP cell sorter (Beckman Coulter, Fullerton, CA). Purified 1 x 105 GFP+ c-kit+ cells were transplanted by tail-vein injection into lethally irradiated (11 Gy total body irradiation) C57BL/6 recipient mice. Reconstituted mice were maintained in a specific pathogen-free environment with acidic water.
Flow cytometric analysis
An IEL cell suspension was prepared as described previously (9). The cells were incubated with mAb for 30 min at 4°C. The following mAb were used for flow cytometry analysis: phycoerythrin-conjugated anti-CD4 (RM4-5), anti-TCR
(GL3) and anti-CD3 (145-2C11), CyChrome-conjugated anti-CD8
(53-6.7), and biotin-conjugated anti-CD8ß (53-5.8), anti-TCR
ß (H57-597) and anti-CD103 (
4ßE) (2E7). All mAb were purchased from PharMingen (San Diego, CA). Biotin-conjugated antibodies were developed with Red 613-conjugated streptavidin (Invitrogen, Carlsbad, CA). Cells from reconstituted mice were gated on GFP+ and data were collected on Epics XL/XL-MCL System II (Beckman Coulter).
Chemotaxis assay
Chemotaxis assays were performed using a 96-well chemotaxis chamber (Neuroprobe, Pleasanton, CA) with a polycarbonate filter (5 µm pore size). Cells were suspended at a density of 1 x 106 /ml in RPMI 1640 medium containing 20 mM HEPES, pH 7.2 and 0.5% BSA (Sigma). Then 25 µl of cell suspension was added into the upper chambers and diluted chemokines (final volume 29 µl) were added into the lower chambers. Chemotaxis chambers were incubated for 2 h at 37°C in 5% CO2.
The numbers of GFP+ migrating cells were counted by Epics Elite ESP cell sorter (Beckman Coulter).
Immunohistochemistry
Longitudinally opened small intestine
10 mm length from reconstituted mice were embedded in OCT compound (Tissue-Tek; Miles, Elkhart, IN) and frozen in liquid nitrogen. The tissue segments were sectioned by a cryostat into 6-µm section, air dried and fixed for 10 min in acetone. The sections were preincubated with Block-ace (Dainippon Pharma ceutical, Osaka, Japan) to inhibit non-specific binding of mAb. The sections were incubated either with isotype-matched biotin-conjugated rat IgG or biotin-conjugated CD8
or anti-c-kit mAb (ACK-2) which was kindly provided by Dr T. Sudo (Toray Industries, Kamakura, Japan) or anti-CD11c (N418; Serotec, Raleigh, NC) for 60 min at room temperature and rinsed with PBS, followed by incubation with peroxidase-conjugated streptavidin (Nichirei, Tokyo, Japan) or peroxidase-conjugated anti-rat IgG (Biosource, Camarillo, CA) or peroxidase-conjugated anti-hamster IgG. Bounded peroxidase activity was visualized with Vectastain AEC substrate kit (Vector, Burlingame, CA) according to the manufacturers instructions. After visualization with substrate, the slides were counterstained with Mayers hematoxylin. Endogenous peroxidase activity was blocked with 0.2% H2O2 and 0.05% NaN3 in distilled water for 10 min at room temperature.
Immunofluorescence analysis
Tissue cryostat sections were preincubated with Block-ace and then incubated with anti-c-kit or biotin-conjugated anti-mCCL25 (R & D Systems, Minneapolis, MN) antibody for 60 min at room temperature, followed by incubation with phycoerythrin-conjugated goat anti-rat IgG (Fab')2 (Southern Biotechnology Associates, Birmingham, AL) or Alexa Fluor 568streptavidin (Molecular Probes, Eugene, OR). Subsequently, the sections were incubated with anti-CD11c mAb and counterstained with Alexa Fluor 488-goat anti-hamster IgG (H + L) (Molecular Probes). For other staining, the sections incubated with biotin-conjugated anti-mCCL25 antibody for 60 min at room temperature, followed by incubation with Alexa Fluor 568streptavidin were counterstained with FITC-conjugated anti-c-kit mAb. Finally, the sections were analyzed by a fluorescence microscope AX-80 (Olympus, Tokyo, Japan).
Cell sorting
Lin cells from BM of 4-week-old C57BL/6 mice were prepared using the combination of biotin-conjugated mAb to lineage (CD3, B220, CD11b, Gr-1, NK1.1 and TER119) and streptavidin-conjugated Microbeads in the MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). The Lin cell population was stained with FITC-conjugated anti-c-kit mAb and c-kit+ Lin cells were sorted by a cell sorter. These highly purified cells were subjected to chemotactic analysis and semiquantitative RT-PCR.
Semiquantitative RT-PCR
Total RNA was prepared from sorted cells using Trizol Reagent (Invitrogen), according to the manufacturers instructions. RNA samples were treated with DNase I to remove contaminating genomic DNA. First-strand cDNA was synthesized at 37°C for 1 h from serially diluted samples in 20 µl of reaction mixture using random primers. Thereafter, cDNA was amplified using a Titanium Taq PCR kit (Clontech, Palo Alto, CA), and the touchdown PCR profile consisted of a 5 min denaturation at 96°C; 10 cycles of 30 s at 96°C and 80 s at 68°C; 25 cycles of 30 s at 96°C and 80 s at 65°C; and a final 5-min extension step at 68°C. The primers for ß-actin were described previously (9). The primers for CCR9 were: 5'-AGGCCAAGAAGTCATCCAAGC-3' and 5'-CCTTCGGAATC TCTCGCCAA-3'. The PCR products were fractionated on 1.5% agarose gel and visualized by ethidium bromide staining.
Statistical analysis
Results of experimental studies are reported as mean ± SD. Differences were analyzed using Students t-test.
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Results
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Generation and analysis of CCL25-IK-transduced mice
A bicistronic retroviral expression vector is shown in Fig. 1(A). Two months after transplantation, high levels of reconstitution with GFP+ cells were observed in the thymus and spleen (Fig. 1B), and also in the BM and peripheral blood (data not shown), in both of GFP- and CCL25-IK-transduced mice. To analyze whether overexpression of the CCL25-IK gene results in selective inhibition of CCR9 expression, we examined the chemotactic response of thymocytes from reconstituted mice to CCL25 using in vitro chemotaxis assays. The response of GFP+ thymocytes from CCL25-IK-transduced mice to CCL25 was dramatically decreased compared with those from control GFP-transduced mice, while the response of both GFP+ thymocytes and splenocytes to CXCL12 was normal (Fig.1 C). These results confirmed specific down-regulation of CCR9 expression in CCL25-IK-transduced mice.

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Fig. 1. Generation of CCL25-IK-transduced mice. (A) Diagrams of bicistronic retroviral expression vector constructs. LTR = long terminal repeat. (B) Reconstitution efficiency in retroviral-transduced mice. Flow cytometric analysis showed GFP expression in thymocytes and splenocytes at 2 months after transplantation with retroviral-transduced c-kit+ cells in irradiated mice. (C) Effect of overexpressed CCL25-IK gene on the function of CCR9 in reconstituted mice. Thymocytes or splenocytes from GFP (open columns) or CCL25-IK (closed columns) gene-transduced mice were stimulated with the indicated concentrations of CCL25 or CXCL12 in a 96-well chemotaxis chamber. The assay was performed in duplicate and the number of migrated GFP+ cells was counted by flow cytometry. Each point represents mean values ± SE from five separate experiments. Statistical analysis was performed by Students t-test (*P < 0.01).
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Down-regulation of CCR9 did not affect thymus-dependent T lymphocyte development
Based on reports describing high expression of CCL25CCR9 in the thymus (1117), we first analyzed GFP+ cell numbers and compositions in the thymi of reconstituted mice by flow cytometry. There were no differences in total GFP+ cell number (Fig. 2A), and the percentages of double-negative, double-positive, CD4 single-positive and CD8 single-positive thymocytes between CCL25-IK- and GFP-transduced mice (data not shown). Thus, these results indicated that CCL25CCR9 is not essential for the development of thymocytes.

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Fig. 2. Flow cytometric analysis in CCL25-IK-transduced mice. (A) Total GFP+ cell numbers of thymus, spleen and IEL of reconstituted mice are shown. The mean values ± SE for eight independent experiments are shown. Statistical analysis was performed by Students t-test (*P < 0.01). (B) The absolute numbers of the indicated GFP+ lymphocytes subsets are shown for IEL from GFP (open columns) or CCL25-IK (closed columns) gene-transduced mice.
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Impaired generation of thymus-independent IEL and CP in CCL25-IK-transduced mice
In contrast, CCL25-IK-transduced mice exhibited a significant reduction in total numbers of GFP+ lymphocytes in the IEL as compared with GFP-transduced mice (Fig.2 A). Flow cytometry analysis revealed a dramatic reduction in the number of various subsets of GFP+ IEL, including CD8
, CD4/CD8, TCR
ß, TCR
and CD3/
Eß7, in the small intestine from CCL25-IK-transduced mice (Fig.2 B). There was no difference in TCR
ß cells in the thymus and spleen between CCL25-IK- and GFP-transduced mice. The total cell numbers of TCR
ß cells in the thymus in GFP- and CCl25-IK-transduced mice were 2.76 ± 0.42 and 2.90 ± 0.23 x 106 respectively (n = 4). Those in the spleen were 1.35 ± 0.12 and 1.40 ± 0.08 x 106 respectively (n = 4). Furthermore, a similar drastic reduction of CD8+ IEL was observed by immunohistochemical analysis of the small intestine (Fig. 3A). These defects were not caused by thymus-dependent T cell development, since flow cytometry analysis of CD8
ß IEL in the small intestine (Fig. 2B) and lymphocytes in the thymus and spleen as well as BM and mesenteric lymph nodes (data not shown) did not show any significant differences between CCL25-IK- and GFP-transduced mice.

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Figure 3. Immunohistological analysis of small intestine and CP, and flow cytometric analysis of IEL in CCL25-IK-transduced mice. (A) Representative immunohistochemical visualization of CD8+ IEL in the small intestine of GFP- and CCL25-IK-transduced mice (x100). The results are from one representative experiments out of five. (B) Representative immunohistochemical visualization of (a, b, d and e) c-kit+, and (c and f) CD11c+ cells in the CP and IEL in the small intestine of (a, b, and c) GFP- and (d, e and f) CCL25-IK-transduced mice. Black arrows indicate c-kit+ cells in the CP: (a) and (e) x20; (b), (c), (e) and (f) x200. The results are from one representative experiments out of three. (B) Flow cytometric profiles of GFP+ cells in IEL of GFP- and CCL25-IK-transduced mice. The results are from one representative experiment out of five.
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These results lead to the question as to whether or not the numbers and generation of CP are normal in the small intestine of CCL25-IK-transduced mice since CP are considered to be the extrathymic anatomical sites for IEL generation (7,8).
In immunohistochemical analysis, it was demonstrated that the numbers and size of CP were significantly reduced, and that the numbers of c-kit+ cells in CP were also very low in CCL25-IK-transduced mice compared with those in GFP-transduced mice (Fig. 3B). However, significant numbers of CD11c+ dendritic cells were still present in CCL25-IK-transduced mice (Fig. 3B). Intriguingly, flow cytometry analysis revealed that the expression level of GFP was significantly decreased in the IEL of CCL25-IK-transduced mice (Fig. 3C). These results suggest that migration of precursor cells from BM into CP was inhibited in the CCL25-IK mice.
Identification of CCL25-producing cells in the CP
We also performed a double immunofluorescence analysis to identify the CCL25-producing cells in the small intestine using 4-week-old wild-type mice.
c-kit+ and CD11c+ cells in CP were present as discrete non-overlapping cell populations in normal wild-type mice as described previously (Fig. 4A) (2).

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Fig. 4. Double immunofluorescence analysis of CP in 4-week-old wild-type mice. (A) Anti-c-kit/anti-CD11c double labeling of CP. Lymphoid progenitor cells are positively stained with anti-c-kit mAb (phycoerythrin), but not with anti-CD11c mAb (FITC), resulting in a red color. Conversely, dendritic cells are positively stained with anti-CD11c mAb, but not with anti-c-kit mAb, resulting in a green color (x400). The results are from one representative experiment out of five. Anti-CCL25/anti-c-kit antibody double labeling of CP. Dendritic cells and intestinal enterocytes are positively stained with anti-CCL25 antibody (phycoerythrin), but not with anti-c-kit mAb (FITC), resulting in a red color (x400). The results are from one representative experiments out of five. (B) Anti-CCL25/anti-CD11c antibody double labeling of CP. Dendritic cells are positively stained with anti-CCL25 antibody (phycoerythrin) followed by anti-CD11c mAb (FITC), resulting in a yellow color, after merging. White arrows indicate double-positive dendritic cells in the CP (x400). The results are from one representative experiment out of five.
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CCL25+ cells which surrounded c-kit+ cells in CP (Fig.4 B) overlapped with CD11c+ dendritic stromal cells (Fig. 4C). During the organogenesis of CP, CD11c+ cells first appear in the CP anlage of the small intestine and form small aggregates (9). Thereafter, c-kit+ cells accumulate in the CP anlage and finally differentiate into TCR+ IEL (8.9). Therefore, this suggested that CCL25 produced by CD11c+ cells in the CP might induce the migration of c-kit+ lympho-hematopoietic progenitors from BM into CP.
CCL25 is an efficacious chemoattractant of c-kit+ lympho-hematopoietic progenitors in BM
To test this hypothesis, we isolated c-kit+ Lin cells from BM of wild-type mice and examined their chemotactic response to CCL25 in vitro. Chemotactic analysis showed that CCL25 possessed chemotactic activity for the c-kit+ Lin cell population in BM equal to or higher than that of CXCL12 (Fig. 5A). However, c-kit Lin+ BM cells showed marginal responses to CCL25 (Fig. 5A). Moreover, RT-PCR analysis revealed that CCR9 mRNA was expressed in a c-kit+ Lin BM cell population (Fig. 5B). These results suggest that CCL25 is involved in the migration of CCR9+ c-kit+ Lin cells into CP.

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Fig. 5. Chemotactic responses of c-kit+ Lin BM cells from C57BL/6 mice (4-weeks-old) to CCL25. (A) c-kit+ Lin cells were highly purified by a cell sorter and subjected into chemotactic analysis. The c-kit+ Lin or c-kit Lin+ cells were stimulated with the indicated concentration of CCL25 (open columns) or CXCL12 (closed columns) in a 96-well chemotaxis chamber. The assay was performed in duplicate and the number of migrated cells was counted by flow cytometry. Each point represents mean values ± SE from three independent experiments. *P < 0.01. (C) Semiquantitative RT-PCR analysis of CCR9 mRNA levels in c-kit+ Lin BM cells, and double-negative and double-positive thymocytes from C57BL/6 mice. Serial 5-fold dilutions of RNAs equivalent to RNAs extracted from the indicated cell number were reverse transcribed, and the cDNA products were amplified by PCR using specific primers, electrophoresed on agarose gels and visualized with ethidium bromide. CCR9 expression was similarly detected in c-kit+ Lin BM cells as well as double-negative and double-positive thymocytes. Note that ß-actin-specific mRNA levels were comparable in all RNA preparations. The results are from one representative experiment out of four.
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Discussion
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Our study demonstrated that reconstituted mice with BM expressing the CCL25-IK gene lacked the responsiveness to CCL25, but not CXCL12.
These reconstituted mice exhibited a dramatic reduction of numbers and average size of CP thereby resulted in a significant reduction of the total cell number of GFP+
ß/
IEL in the small intestine. Furthermore, we showed the existence of CCL25-expressing CD11c+ dendritic cells in CP and the possible involvement of such CD11c+ cells in recruiting c-kit+ Lin/CCR9+ cells from BM into CP.
Recently, another group reported the phenotype of CCR9-deficient mice (21). They detected a decrease in the IEL:epithelial cell ratio and percentage of TCR
CD8
+ IEL, and moderate increase of TCR
cells in the spleen and lymph nodes of CCR9-deficient mice. From these results, they speculated that TCR
cells in the spleen and lymph nodes of CCR9-deficient mice lack the ability to migrate into the intestinal epithelium. However, they did not mention the CP or selective defects of the appearance of IEL from extrathymic origin in the CCR9-deficient mice. In our study, we detected significant reduction of various subsets of IEL (CD8
, CD4/CD8, TCR
ß, TCR
and CD3/
Eß7). These defects were probably caused by the thymus-independent T cell development since we detected a dramatic reduction of the total numbers and size of CP in the CCL25-IK-transduced mice, whereas no significant differences in total cell number and cellularity in the thymus, spleen, mesenteric lymph nodes, BM and even CD8
ß+ IEL in the small intestine of these mice.
Furthermore, they detected a 1-day delayed appearance of double-positive thymocytes of CCR9-deficient mice in fetal ontogeny (21), but it is difficult to analyze the effect of the CCL25-IK gene on the function of CCR9 in the fetal stage using our system. Based on these results, it is very possible that reduction of IEL in the CCL25-IK-transduced mice was caused by the impaired formation of CP.
In contrast to CP, molecular mechanisms of PP organogenesis have been extensively studied (2224). PP and peripheral lymph nodes are absent from lymphotoxin-deficient and aly/aly mutant mice, whereas CP and
ß/
-IEL are present in these mice (25,26). A chemokine receptor CXCR5 and its ligand interaction CXCR5CXCL13 (BLC) have been shown to be required for the initial step of PP organogenesis (2729). In addition, a profound morphological alteration of all secondary lymphoid organs including PP was observed in mice that lacked CCR7CCL19 (ELC) signaling, although CCR7-deficient mice develop normal numbers of PP (30).
These results clearly demonstrate distinct pathways in CP formation from PP organogenesis. At present, cytokine CR
chain is the only molecule reported to be indispensable for CP organogenesis since CR
-deficient mice lack both CP and thymus-independent IEL (10). Our study revealed that the formation of CP and consequent appearance of
ß/
-IEL were severely impaired in reconstituted mice with BM expressing the CCL25-IK gene. Furthermore, we showed the existence of CCL25-expressing CD11c+ dendritic cells in CP and suggested the role of such CD11c+ cells in recruiting c-kit+ Lin/CCR9+ cells from BM into CP.
In conclusion, our study has established a novel function of CCL25CCR9 in the formation of CP and consequent appearance of IEL in the mouse small intestine. We propose the following provisional scenario for organogenesis of CP. CD11c+ dendritic cells first migrate, perhaps, from BM into the crypt of lamina propria, give rise to a microenvironment of CP anlagen by forming a cell aggregate and start to produce CCL25 (I). Then, c-kit+ Lin/CCR9+ lympho-hematopoietic progenitors in BM are recruited into the CP anlagen (II) and eventually, these progenitor cells differentiate into c-kit+ TCR precursor IEL in the histologically matured CP (III). Finally, these c-kit+ TCR CP cells move into the intestinal epithelium possibly also via CCL25CCR9 interaction and differentiate into mature TCR+ IEL in the epithelial microenvironment in situ (9).
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Acknowledgements
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We are very grateful to Dr J. J. Oppenheim (National Cancer Institute, Frederick, MD) for pre-review of this study. The present study was supported by the CREST research project of Japan Science and Technology Corp.
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Abbreviation
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BMbone marrow
CPcryptopatch
CRcommon receptor
GALTgut-associated lymphoid tissue
GFPgreen fluorescent protein
IELintraepithelial T lymphocytes
IKintrakine
IRESinternal ribosomal entry site
PPPayers patch
SCFstem cell factor
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