Development and Migration a Role for CCR9 in T Lymphocyte

A Role for CCR9 in T Lymphocyte Development and Migration Shoji Uehara, Alexander Grinberg, Joshua M. Farber and Paul E. Love This information is current as of October 2, 2021. J Immunol 2002; 168:2811-2819; ; doi: 10.4049/jimmunol.168.6.2811 http://www.jimmunol.org/content/168/6/2811 Downloaded from References This article cites 47 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/168/6/2811.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. A Role for CCR9 in T Lymphocyte Development and Migration1

Shoji Uehara,* Alexander Grinberg,* Joshua M. Farber,† and Paul E. Love2*

CCR9 mediates chemotaxis in response to CCL25/thymus-expressed chemokine and is selectively expressed on T cells in the thymus and small intestine. To investigate the role of CCR9 in T cell development, the CCR9 gene was disrupted by homologous recombination. B cell development, thymic ␣␤-T cell development, and thymocyte selection appeared unimpaired in adult CCR9- deficient (CCR9؊/؊) mice. However, competitive transplantation experiments revealed that bone marrow from CCR9؊/؊ mice was less efficient at repopulating the thymus of lethally irradiated Rag-1؊/؊ mice than bone marrow from littermate CCR9؉/؉ mice. CCR9؊/؊ mice had increased numbers of peripheral ␥␦-T cells but reduced numbers of ␥␦TCR؉ and CD8␣␤؉␣␤TCR؉ intraepithelial lymphocytes in the small intestine. Thus, CCR9 plays an important, although not indispensable, role in regulating .the development and/or migration of both ␣␤؊ and ␥␦؊ T lymphocytes. The Journal of Immunology, 2002, 168: 2811–2819 Downloaded from evelopment of T cells continues into adulthood and re- both cortex and medulla (10, 11). Interestingly, CCL25 is also quires the periodic migration of progenitor cells from the expressed in fetal thymus, raising the possibility that it may par- D bone marrow to the thymus (1, 2). The ordered progres- ticipate in recruiting T progenitor cells to the thymus (12). The sion of thymocytes through distinct stages of development is also only other known site of CCL25 production is the epithelial layer associated with the migration of cells into and between different of the small intestine (11, 13–15). Thus, CCL25 may also be im- thymic microenvironments (3). Chemokines are a group of small portant for the development, homeostasis, and/or function of mu- http://www.jimmunol.org/ (8–14 kDa), mostly basic, structurally related molecules that reg- cosal T lymphocytes. ulate trafficking of leukocytes through interactions with a subset of CCR9 mediates chemotaxis in response to CCL25 (16–19). seven-transmembrane, G protein-coupled receptors (4–8). Several CCR9 is expressed on the majority of immature CD4ϩCD8ϩ (dou- different chemokines are expressed in the thymus, including ble-positive (DP)) thymocytes, is down-regulated during their tran- XCL1/lymphotactin, CCL3/macrophage-inflammatory protein sition to the CD4ϩ or CD8ϩ (single-positive (SP)) stage, and is 3 ␣ ␤ (MIP) -1 , CCL4/MIP-1 , CCL17/thymus and activation-regu- expressed on a minor subset of CD8ϩ lymph node T cells (20, 21). lated chemokine, CCL19/EBI-1 ligand chemokine/MIP-3␤, ϩ CD69 thymocytes demonstrate enhanced CCL25-induced migra- CCL21/6-cysteine chemokine/secondary lymphoid-tissue chemo- Ϫ

tion compared with CD69 thymocytes (21, 22), and thymocyte by guest on October 2, 2021 kine, CCL25/thymus-expressed chemokine, and CXCL12/stromal migration in response to CCL25 is augmented by TCR signaling cell-derived factor-1, suggesting that chemokines may play an im- (21). These findings suggest that CCR9 may be involved in regu- portant role in thymopoiesis (4–8). Although a great deal has been lating T cell migration within the thymus, particularly during thy- learned about the function of chemokines and chemokine receptors mocyte selection. Approximately half of all ␥␦TCRϩ thymocytes in regulating the migration of mature lymphocytes, little informa- ␥␦ tion is available concerning their potential role in lymphocyte de- and peripheral -T cells express CCR9, and these cells migrate to ␥␦ velopment. CCL25 (21). The expression of CCR9 on specific -T cell subsets ␥ ϩ ␥ ϩ Expression of CCL25/thymus-expressed chemokine was ini- (e.g., V 2 , but not V 3 ) indicates that CCR9 may also function ␥␦ tially detected in medullary dendritic cells (9), but recent experi- in the development and/or trafficking of -T cells (21). Finally, ments indicate that it is also expressed by thymic epithelial cells in pre-pro-B cells in the bone marrow respond to CCL25, raising the possibility that CCR9 may regulate the early stages of B cell development (23). *Laboratory of Mammalian Genes and Development, National Institute of Child To investigate the role of CCR9 during lymphocyte develop- † Health and Human Development, and Laboratory of Clinical Investigation, National Ϫ/Ϫ Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, ment, we generated CCR9-deficient (CCR9 ) mice by homol- MD 20892 ogous recombination. Surprisingly, both ␣␤-T cell and B cell de- Ϫ/Ϫ Received for publication November 27, 2001. Accepted for publication January velopment appeared normal in adult CCR9 mice. In addition, 18, 2002. thymocyte selection of ␣␤-lineage T cells was unaffected in The costs of publication of this article were defrayed in part by the payment of page CCR9Ϫ/Ϫ mice, even through most DP thymocytes express CCR9 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. at high levels and respond to CCL25. However, the results of com- 1 This work was supported in part by an Intramural AIDS Targeted Antiviral Program petitive bone marrow transplantation experiments demonstrated Ϫ Ϫ grant. S.U. is supported by Japan Society for the Promotion of Science Research that CCR9 / bone marrow cells had a reduced capacity to re- Fellowships for Japanese Biomedical and Behavioral Researchers at the National populate the thymus compared with bone marrow cells from Institutes of Health. CCR9ϩ/ϩ mice. These results suggest that CCR9 may be in- 2 Address correspondence and reprint requests to Dr. Paul E. Love, Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human volved in regulating the migration of progenitor cells to the Development, National Institutes of Health, Bethesda, MD 20892. E-mail address: thymus or the retention of T progenitor cells in the thymus. [email protected] Ϫ Ϫ CCR9 / mice also contained increased numbers of peripheral 3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; DN, dou- ␥␦ ␥␦ ϩ ble negative; DP, double positive; IEL, intraepithelial lymphocyte; TN, triple nega- -T cells but reduced numbers of TCR intraepithelial lym- tive; ES cell, embryonic stem cell. phocytes (IEL) in the small intestine. Thus, CCR9 also plays an

important role in regulating the development and/or migration 107 cell/ml in RPMI 1640 plus 0.5% BSA, and 100 ␮l cell suspension was of ␥␦-T lymphocytes. added to an insert in a well with 600 ␮l medium. After equilibration at 37°C for 1 h chemokines were added to the wells and the plates were incubated for an additional 2 h before cells were harvested, collected by Materials and Methods centrifugation, and counted. Duplicate wells were used for each condition. Generation of CCR9-deficient mice Murine CXCL12 and CCL25 were obtained from PeproTech (Rocky Hill, NJ) and R&D Systems (Minneapolis, MN), respectively. An ϳ15-kb fragment containing the mouse CCR9 gene was cloned from a ␭ 129 SvJ genomic library (Stratagene, La Jolla, CA). A 7-kb EcoRV Northern blotting fragment and a 1.6-kb HindIII-EcoRV fragment were then cloned into the XpPNT (NEO/TK) vector (24). The targeting construct was linearized with Total RNA was isolated from various organs from B6 and Rag-1Ϫ/Ϫ mice NotI and electroporated into 2 ϫ 107 embryonic stem cells (ES cells). After using TRIzol (Life Technologies, Gaithersburg, MD). Twenty micrograms transfection, ES cells were selected in the presence of G418 and gancy- of total RNA was fractionated on a 1% agarose/formaldehyde gel and clovir and screened for homologous recombination. Chimeric mice were transferred onto a GeneScreen Plus nylon membrane (New England Nu- generated from CCR9ϩ/Ϫ ES cell clones by injection into B6 blastocysts, clear, Boston, MA). The membrane was hybridized with 32P-labeled cDNA and germline transmission of the mutant allele was confirmed by Southern fragments encoding mouse CCR9, mouse CCL25, or human EF-1␣. blot analysis of DNA obtained from tail biopsies. H-Y (25), P14 (26), and AND (27) ␣␤TCR transgenes were bred into Bone marrow chimeras the CCR9Ϫ/Ϫ background. The phenotype of thymocytes and peripheral T ϩ/ϩ cells was analyzed by staining with PE-labeled anti-CD4, CyChrome-la- Bone marrow cells were isolated from femurs of CCR9 (CD45.2), Ϫ/Ϫ a beled anti-CD8␣, and FITC-labeled anti-H-Y clonotypic receptor (T3.70), CCR9 (CD45.2), and B6.SJL-Ptprc /BoAiTac mice (B6.CD45.1; Tac- 6 anti-TCR-V␣ 2 (P14), or anti-TCR-V␣ 11 (AND) mAb as previously de- onic Farms, Lexington, KY). A total of 2 ϫ 10 cells consisting of various ϩ/ϩ scribed (28). Mice were maintained in the H-2Db background by mating ratios of CCR9 (CD45.2) and B6.CD45.1 bone marrow cells or Ϫ/Ϫ with C57BL/6 mice. CCR9 (CD45.2) and B6.CD45.1 bone marrow cells were injected i.v. Downloaded from into lethally irradiated (9.5 Gy) Rag-1Ϫ/Ϫ B6 mice (CD45.2). One to 2 mo Cell preparation and flow cytometry after the transplantation thymocytes and lymph node B cells were analyzed by FACS for the presence of CD45.2ϩ cells. Thymus, spleen, lymph node, and bone marrow were excised from mice, and single-cell suspensions were prepared. IEL were prepared from small Statistical analysis and large intestines according to conventional methods (29). Standard flow cytometry was performed as previously described using a FACSCalibur Data from mice of the different phenotypes were analyzed using Student’s and CellQuest software (BD Biosciences, Mountain View, CA) (21). Anti- t test. http://www.jimmunol.org/ CD3⑀, anti-CD4, anti-CD8␣, anti-CD8␤, anti-CD25, anti-CD19, anti- CD44, anti-B220, anti-CD45.2, anti-␤TCR, anti-␦TCR, anti-TCR-V␥2, Results anti-TCR-V␦4, anti-TCR-V␣2, anti-TCR-V␣11, and anti-DX5 mAb were purchased from BD PharMingen (San Diego, CA). The polyclonal anti- Generation of CCR9-deficient mice CCR9 Ab has been described previously (21). Anti-H-Y clonotypic recep- To investigate the biological significance of CCR9 in vivo, tor mAb (T3.70) was purified from cell culture supernatants in our labo- Ϫ/Ϫ ratory. Anti-TCR-V␥1 (2.11) (30) and anti-TCR-V␥5 (GL1) (31) mAbs CCR9 mice were generated by homologous recombination. were provided by Dr. L. Lefrancois (University of Connecticut Health The gene-targeting vector was designed to delete exon 4, which Center, Farmington, CT). Unconjugated anti-Fc␥RII (2.4G2) was used to contains most of the CCR9-coding sequence (Fig. 1A). The ex- block nonspecific binding of the labeled Ab. PE-conjugated streptavidin pected structure of the targeted CCR9 locus was confirmed by by guest on October 2, 2021 and CyChrome-conjugated streptavidin were also purchased from BD Southern blotting with the indicated 3Ј probe (Fig. 1B). CCR9 PharMingen. mRNA was undetectable in the thymus, spleen, lymph node, and Chemotaxis assays small intestine from CCR9Ϫ/Ϫ mice (data not shown). FACS analysis confirmed that thymocytes from CCR9Ϫ/Ϫ mice lacked CCR9 Chemotaxis assays were performed as previously described (18, 21) with Ϫ/Ϫ modifications, using 6.5-mm Transwell tissue culture inserts with a 5-␮m surface expression (Fig. 1C). CCR9 mice did not exhibit any pore size (Corning, Cambridge, MA). Thymocytes were suspended at 1 ϫ developmental abnormalities, were produced in Mendelian ratios,

FIGURE 1. Targeted disruption of the murine CCR9 gene. A, Targeting vector and homologous recombination at the CCR9 locus. Top, Restriction map of the murine CCR9 locus. Middle, The CCR9 gene targeting construct. An EcoRI- HindIII fragment containing most of the exon 4 coding sequence was replaced with a phosphoglyceraldehyde kinase-directed neomycin cassette (Neo). Bottom, Pre- dicted structure of the disrupted CCR9 gene. B, Southern blot analysis of genomic DNA. DNA from mouse tails was digested with HindIII and probed with the 3Ј probe depicted in A. C, FACS analysis of CCR9 surface expression on thymocytes. Total thymocytes from CCR9ϩ/ϩ, CCR9ϩ/Ϫ, and CCR9Ϫ/Ϫ mice were incubated with biotinylated anti-CCR9 Ab (open tracing) or biotinylated control rabbit Ig (shaded tracing), then labeled with PE-conjugated streptavidin and analyzed by FACS. The Journal of Immunology 2813 and were indistinguishable from CCR9ϩ/ϩ or CCR9ϩ/Ϫ littermates on the basis of size, activity, or fertility (data not shown).

Normal ␣␤-lineage T cell development in adult CCR9Ϫ/Ϫ mice We initially focused our analysis on T cell development in CCR9Ϫ/Ϫ mice, because CCR9 is expressed on the surface of most thymocytes, especially CD4ϩCD8ϩ (DP) cells (20, 21). The numbers and distribution of the major thymic and peripheral T cell subsets, as deﬁned by the expression of CD4 and CD8, were normal in CCR9Ϫ/Ϫ mice (Fig. 2). In addition, surface expression of CD3, ␤TCR, CD5, and CD69 were indistinguishable on thymocytes and lymph node T cells from CCR9Ϫ/Ϫ and CCR9ϩ/ϩ littermates (data not shown). Histological studies revealed normal thymus architecture in CCR9Ϫ/Ϫ mice (data not shown). Periph- eral T cells from CCR9Ϫ/Ϫ mice demonstrated normal prolifera- Ϫ/Ϫ tive responses after CD3 cross-linking, and CCR9 mice had FIGURE 3. Lack of chemotactic response of thymocytes from normal Ab responses to T cell-dependent Ags (data not shown). CCR9Ϫ/Ϫ mice to CCL25. Total thymocytes from CCR9ϩ/ϩ, CCR9ϩ/Ϫ, Finally, there were no differences in the number or phenotype of and CCR9Ϫ/Ϫ mice were used for chemotaxis assays with CCL25 (200 Ϫ/Ϫ ϩ/ϩ nM) and CXCL12 (50 nM). Results are expressed as cells migrating per

thymocytes from newborn CCR9 and CCR9 mice (data not Downloaded from shown). Collectively, these data indicate that ␣␤-T cells can de- 106 input cells. Determinations were performed in duplicate, and error bars velop normally in the absence of CCR9. represent the SD. We next analyzed the responsiveness of thymocytes to CCL25. CCR9Ϫ/Ϫ thymocytes did not respond to CCL25, although they migrated to CXCL12 in a manner comparable to CCR9ϩ/ϩ cells or the AND-TCR transgene (data not shown). These data suggest (Fig. 3). CCR9ϩ/Ϫ thymocytes expressed CCR9 at lower levels that CCR9 does not play a critical role in thymocyte selection. http://www.jimmunol.org/ than CCR9ϩ/ϩ littermates (Fig. 1C) and exhibited a reduced che- Ϫ/Ϫ ␥␦ motactic response to CCL25 (Fig. 3). These data indicate that CCR9 mice contain increased numbers of -T lymphocytes CCR9 expression is essential for chemotaxis of thymocytes to in spleen and lymph node CCL25. Approximately half of all ␥␦TCRϩ cells in thymus, spleen, and CCL25 preferentially induces the migration of CD3highCD69ϩ lymph node express CCR9 and can respond to CCL25 (21). There- DP thymocytes compared with CD69Ϫ DP thymocytes (20–22), fore, we next compared the number and phenotype of ␥␦-T cells in and TCR cross-linking of DP thymocytes enhances CCL25-medi- different organs from CCR9Ϫ/Ϫ and CCR9ϩ/ϩ mice. The number ated migration (21). These data suggest that CCL25 may be in- and percentage of ␥␦TCRϩ thymocytes were similar in CCR9Ϫ/Ϫ ϩ/ϩ volved in thymocyte selection. To determine whether positive or and CCR9 mice (Fig. 4A). However, both the absolute num- by guest on October 2, 2021 negative selection was affected in CCR9Ϫ/Ϫ mice, we crossed the ber and the percentage of ␥␦TCRϩ cells were increased ϳ2-fold in H-Y TCR transgene into the CCR9Ϫ/Ϫ background and analyzed spleen and lymph nodes of CCR9Ϫ/Ϫ mice compared with the phenotype of thymocytes in the positively selecting (female) or CCR9ϩ/ϩ mice (Fig. 4A). We previously observed that ␥␦TCRϩ negatively selecting (male) background. The absence of CCR9 did cells that resemble recent thymic emigrants (i.e., not alter the efficiency of either positive or negative thymocyte CD44lowCD45RBlow) preferentially express CCR9 (21). However, ␣ selection in H-Y TCR transgenic mice (data not shown). In addi- no significant differences in CD44, CD45RB, CD62L, or IEL in- tion, no differences in the efficiency of thymocyte selection were tegrin expression were observed on peripheral ␥␦-T cells from observed in CCR9Ϫ/Ϫ mice that expressed the P14-TCR transgene CCR9Ϫ/Ϫ and CCR9ϩ/ϩ mice (data not shown). To ascertain

FIGURE 2. Normal development of ␣␤-T cells in adult CCR9Ϫ/Ϫ mice. A, Rep- resentative two-color ﬂuorescence plots showing the expression of CD4 and CD8␣ on thymocytes and lymph node cells from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice. B, Compar- ison of total cell numbers from thymus, lymph node, and spleen from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice. C, Frequency of CD4Ϫ CD8Ϫ (DN), CD4ϩCD8ϩ (DP), CD4ϩ CD8Ϫ (CD4-SP), and CD4ϪCD8ϩ (CD8- SP) cells in thymus from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice. D, Frequency of CD3ϩ, CD4ϩ, CD8␣ϩ, and B220ϩ cells in lymph nodes from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice. Open and closed circles represent data from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice, respectively. 2814 CHARACTERIZATION OF CCR9-DEFICIENT MICE

from CCR9Ϫ/Ϫ mice resembled those from CCR9ϩ/ϩ mice in that ␣ϩ ␣ they were uniformly CD8 and expressed high levels of IEL integrin (data not shown). Although the TCR-V␥/V␦ repertoire is diverse in ␥␦TCRϩ IEL, the ␥␦TCRs expressed by small intestinal IEL consist predomi- nantly of TCR-V␥1or-V␥5 paired with TCR-V␦4, -V␦5, -V␦6, or -V␦7 (33, 34). To assess whether there were any differences in the TCR-V␥/V␦ repertoire of ␥␦TCRϩ IEL in small intestine, we analyzed ␥␦TCRϩ IEL in CCR9Ϫ/Ϫ and CCR9ϩ/ϩ mice for the expression of TCR-V␥1, -V␥2, -V␥5, and -V␦4byﬂow cytometry. The percentages of TCR-V␥1, -V␥2, and -V␥5-bearing cells among ␥␦TCRϩ IEL were similar in CCR9Ϫ/Ϫ and CCR9ϩ/ϩ mice (Fig. 5, D and E). However, the percentage of TCR-V␦4- bearing IEL was markedly decreased in CCR9Ϫ/Ϫ mice (Fig. 5, D and E). Most TCR-V␦4ϩ IEL coexpress TCR-V␥5 and, to a lesser extent, TCR-V␥1. Consistent with this observation, both TCR- V␥5/V␦4ϩ and TCR-V␥1/V␦4ϩ IEL subsets were decreased in CCR9Ϫ/Ϫ mice (Fig. 5E). Collectively, these data indicate that CCR9 is involved in the generation or maintenance of ␥␦TCRϩ Downloaded from IEL and, in particular, TCR-V␦4ϩ IEL in small intestine. We next evaluated ␣␤TCRϩ IEL in the small intestine of FIGURE 4. ␥␦-T cells are increased in lymph nodes and spleen in Ϫ/Ϫ ϩ/ϩ ␣␤ ϩ Ϫ/Ϫ CCR9 and CCR9 mice. TCR IEL subsets can be dis- CCR9 mice. A, Thymocytes, lymph node cells, and spleen cells were ␣ ␤ ␦ tinguished by the expression of CD8 , CD8 , and CD4. The per- isolated and stained with mAbs directed to CD3, TCR, and B220. For ϩ ϩ Ϫ ϩ ϩ ϩ␦ ϩ Ϫ centage of ␣␤TCR IEL that were CD4 CD8␣ and CD4 CD8

thymocytes, the percentage of CD3 TCR B220 cells among total cells http://www.jimmunol.org/ Ϫ/Ϫ ϩ/ϩ is shown. For lymph node and spleen, CD3ϩB220Ϫ cells were gated, and was not consistently different in CCR9 and CCR9 mice ϩ the percentage of ␦TCRϩ cells among total CD3ϩ cells is shown. The (Fig. 5F). However, the percentage of CD8␣␤ IEL was reduced ϩ percentage of ␥␦TCR cells was increased in lymph node and spleen of in CCR9Ϫ/Ϫ mice (Fig. 5F). No significant differences were ob- Ϫ/Ϫ ϩ/ϩ CCR9 mice compared with CCR9 mice (p Ͻ 0.001). B, Lymph served in the number or subset distribution of small intestinal lam- ␥ node cells were stained with mAbs directed to CD3, B220, and TCR-V 1, ina propria lymphocytes in CCR9Ϫ/Ϫ and CCR9ϩ/ϩ mice (data not TCR-V␥2, TCR-V␥5, or TCR-V␦4. CD3ϩB220Ϫ cells were gated, and the percentages of TCR-V␥1-, TCR-V␥2-, TCR-V␥5-, or TCR-V␦4-express- shown). ing cells among total CD3ϩ cells are shown. Open and closed circles represent data from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice, respectively. Ϫ/Ϫ Normal B lymphopoiesis in CCR9 mice by guest on October 2, 2021 Northern blot analysis revealed that CCR9 mRNA is expressed in Ϫ Ϫ whether ␥␦-T cell subsets were different in CCR9Ϫ/Ϫ and bone marrow from B6 mice (Fig. 6A). Rag-1 / bone marrow CCR9ϩ/ϩ mice, we analyzed the percentages of TCR-V␥1, -V␥2, cells, which lack mature T and B cells, had an equivalent level of -V␥5, or -V␦4-expressing cells among total CD3ϩ T cells. TCR- CCR9 mRNA expression, indicating that CCR9 is expressed on V␥1ϩ,-V␥2ϩ -V␥5ϩ,or-V␦4ϩ cells appeared to be increased in lymphoid progenitor cells and/or myeloid cells. In contrast to Ϫ Ϫ the lymph node (Fig. 4B) and spleen (data not shown) of CCR9 / CCR9 expression, CCL25 mRNA was undetectable in the bone ϩ ϩ ϩ mice. On average, TCR-V␥1 , TCR-V␥5 , and TCR-V␦4 cells marrow (Fig. 6A). Bowman et al. (23) described an immature pop- ϩ ϩ were increased more than TCR-V␥2 cells (TCR-V␥1 , 3.1-fold; ulation of bone marrow cells that migrates in response to CCL25. ϩ ϩ ϩ TCR-V␥2 , 1.8-fold; TCR-V␥5 , 2.9-fold; TCR-V␦4 , 2.6-fold). This population is phenotypically similar to DX5ϪCD19ϪB220ϩ ␥ ␥ ␥ Ϫ Ϫ ϩ In contrast, no significant difference in TCR-V 1, -V 2, -V 5, or bone marrow cells (35, 36). DX5 CD19 B220 cells can be fur- ␦ Ϫ/Ϫ -V 4 usage was detected in thymocytes from CCR9 and ther subdivided on the basis of CD4 surface expression into B cell ϩ/ϩ CCR9 mice (data not shown). precursors (CD4ϪDX5ϪCD19ϪB220ϩ) and cells of unknown lin- ϩ Ϫ Ϫ ϩ Abnormal distribution of ␥␦TCRϩ IEL in CCR9Ϫ/Ϫ mice eage and potential (CD4 DX5 CD19 B220 ) (37). Signifi- cantly, staining of bone marrow cells with anti-CCR9 Ab revealed Mucosal lymphocytes are composed of IEL and lamina propria ϩ Ϫ Ϫ ϩ ϩ ϩ high level expression of CCR9 on CD4 DX5 CD19 B220 lymphocytes. IEL consist of ␣␤TCR and ␥␦TCR cells, with Ϫ Ϫ Ϫ ϩ cells, but only very low levels of CCR9 on CD4 DX5 CD19 ␥␦TCR cells making up ϳ50% of the total population in the ϩ B220 cells (Fig. 6B). Both populations migrated in response to small intestine and ϳ20% of the total population in the large in- CCL25 (data not shown). No statistically significant differences in testine (32). CCL25 and CCR9 mRNAs are detectable in duode- the number or the percentage of CD4ϩDX5ϪCD19ϪB220ϩ and num and small intestine, but not in esophagus, stomach, appendix, Ϫ Ϫ Ϫ ϩ and large intestine (Fig. 5A). In addition, CCR9 mRNA is ex- CD4 DX5 CD19 B220 bone marrow cells were observed in Ϫ/Ϫ ϩ/ϩ Ϫ/Ϫ pressed in both ␣␤TCRϩ and ␥␦TCRϩ small intestinal IEL (21). CCR9 and CCR9 mice (Fig. 6C). In addition, CCR9 The number of small intestinal IEL or large intestinal IEL was not mice contained normal numbers of bone marrow cells and periph- ϩ statistically different in CCR9Ϫ/Ϫ and CCR9ϩ/ϩ mice (data not eral B cells and contained normal proportions of pro-B (CD43 Ϫ low Ϫ Ϫ low Ϫ shown). However, in CCR9Ϫ/Ϫ mice the percentage of ␥␦TCRϩ IgM B220 ), pre-B (CD43 IgM B220 ), immature B (CD43 ϩ Ϫ ϩ IEL was decreased in small intestine but increased in large intes- IgM B220low), and recirculating bone marrow (CD43 IgM tine (Fig. 5, B and C). There was no difference in CD2 and CD8␣ B220high) B cells and exhibited normal IgM and IgD surface profiles expression on large intestinal IEL from CCR9Ϫ/Ϫ and CCR9ϩ/ϩ on splenic B cells (Fig. 6C and data not shown). Thus, CCR9 is not mice (data not shown). In addition, small intestinal ␥␦TCRϩ IEL essential for normal B cell development. The Journal of Immunology 2815 Downloaded from

FIGURE 5. Reduction in small intestinal IEL in CCR9Ϫ/Ϫ mice. A, Northern blot determination of CCL25 and CCR9 mRNA expression in gastrointestinal tract. Twenty micrograms of total RNA from various tissues was separated by gel electrophoresis, transferred to membranes, and hybridized with http://www.jimmunol.org/ cDNA probes to mouse CCL25, mouse CCR9, or human EF-1␣. Lane 1, thymus; lane 2, esophagus; lane 3, stomach; lane 4, duodenum; lane 5, small intestine; lane 6, appendix; lane 7, large intestine. B, Representative FACS analysis of puriﬁed IEL from small and large intestines. Small and large intestinal IEL were isolated from CCR9ϩ/ϩ or CCR9Ϫ/Ϫ mice. The cells were stained with mAbs to CD3, ␤TCR, and ␦TCR and then analyzed by FACS. CD3ϩ cells were gated and analyzed for ␤TCR and ␦TCR expression. Values indicate the percentage of cells in each quadrant. C, ␥␦TCRϩ IEL are decreased in the small intestine of CCR9Ϫ/Ϫ mice relative to CCR9ϩ/ϩ mice (p Ͻ 0.01). The percentages of ␥␦TCRϩ IEL among CD3ϩ small intestinal IEL were plotted. E, Data from CCR9ϩ/ϩ mice; F, data from CCR9Ϫ/Ϫ mice. D, Reduction in TCR-V␦4ϩ small intestinal IEL in CCR9Ϫ/Ϫ mice relative to CCR9ϩ/ϩ mice (p Ͻ 0.01). Small intestinal IEL from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice were stained with mAbs against CD3, ␦TCR, and TCR-V␥1, TCR-V␥2, or TCR-V␥5. CD3ϩ␦TCRϩ cells were gated, and the percentages of TCR-V␥1-, TCR-V␥2-, and TCR-V␥5-expressing cells were analyzed. Because mAbs directed to ␦TCR and TCR-V␦4 compete for binding, CD3ϩ␤TCRϪ IEL were gated to determine the percentage of TCR-V␦4ϩ cells. E, ϩ ϩ Ϫ Ϫ ϩ ϩ Ϫ Ϫ TCR-V␥1/V␦4 and TCR-V␥5/V␦4 IEL are decreased in CCR9 / mice. Small intestinal IEL from CCR9 / and CCR9 / mice were stained with by guest on October 2, 2021 mAbs directed to CD3, ␤TCR, TCR-V␦4, and TCR-V␥1, or TCR-V␥5. CD3ϩ␤TCRϪ cells were gated and analyzed for TCR-V␦4 and TCR-V␥1or TCR-V␥5 expression. Values indicate the percentage of cells in each quadrant. F, Reduction in ␣␤TCRϩCD8␣␤ϩ small intestinal IEL in CCR9Ϫ/Ϫ mice relative to CCR9ϩ/ϩ mice (p Ͻ 0.05). Small intestinal IEL from CCR9ϩ/ϩ and CCR9Ϫ/Ϫ mice were stained with mAbs directed to ␤TCR, CD4, CD8␣, and CD8␤. ␤TCRϩ cells were gated and analyzed for the percentages of CD8␣␣ (CD8␣ϩCD8␤Ϫ), CD8␣␤ (CD8␣ϩCD8␤ϩ), CD4ϪCD8␣ϩ, CD4ϩCD8␣ϩ, and CD4ϩCD8␣Ϫ cells in ␣␤TCRϩ IEL. E, Data from CCR9ϩ/ϩ mice; F, data from CCR9Ϫ/Ϫ mice.

Reduced thymus repopulating activity of CCR9Ϫ/Ϫ bone whether the loss of CCR9 affects the migration of bone marrow marrow cells progenitor cells into the thymus or the establishment or retention As shown in Fig. 2, no obvious defects in ␣␤-T cell development of T progenitor cells in the thymus, we performed a competitive were observed in adult CCR9Ϫ/Ϫ mice. Bleul et al. (12) reported transplantation experiment. Total bone marrow cells from Ϫ/Ϫ ϩ/ϩ previously that CCL25 is expressed in the early thymic anlage of CCR9 mice (CD45.2) and B6.CD45.1 (CCR9 ) mice were the mouse fetus, and that fetal blood prothymocytes (Thy1ϩc- mixed in different ratios and injected into lethally irradiated Rag- Ϫ/Ϫ kitlow) respond to CCL25. These findings suggest that CCL25/ 1 (CD45.2) mice. As a control, identical mixtures of bone mar- ϩ/ϩ CCR9 may be involved in the migration of prothymocytes into the row cells from littermate CCR9 (CD45.2) and B6.CD45.1 Ϫ/Ϫ thymus. To explore this possibility further, we examined CCR9 mice were injected into irradiated Rag-1 (CD45.2) mice. One expression on immature thymocyte populations from adult mice. to 2 mo after the bone marrow transplantation, the number and ϩ CCR9 expression was undetectable on CD3ϪCD4ϪCD8Ϫ (triple- percentage of CD45.2 thymocytes and peripheral (lymph node) B Ϫ/Ϫ negative (TN)) thymocytes from adult mice, including the most cells were determined. Significantly, when mixtures of CCR9 immature (CD44ϩCD25Ϫ TN) subset, and adult TN thymocytes and B6.CD45.1 bone marrow were injected, the percentage of thy- Ϫ Ϫ cells failed to migrate in response to CCL25 (21) (data not shown). mocytes derived from CCR9 / bone marrow cells was consis- In addition, the number and distribution of TN thymocyte subsets tently lower than the expected ratio (Fig. 7, A and C). In contrast, Ϫ Ϫ in CCR9Ϫ/Ϫ and CCR9ϩ/ϩ mice, as defined by the expression of the percentage of lymph node B cells derived from CCR9 / bone CD44 and CD25, were similar (data not shown). Thymus size and marrow cells was consistently close to the expected ratio (Fig. 7D). Ϫ Ϫ cellularity can be normal even if the number of immature T pro- The reduction in CCR9 / bone marrow-derived thymocytes was genitor cells in the thymus is reduced, presumably because these evident at both the CD44ϩ/ϪCD25ϩ TN and the CD44ϪCD25Ϫ cells are capable of expanding (1). Consequently, examination of TN stage (Fig. 7B), indicating that cells from CCR9Ϫ/Ϫ mice were thymocytes in the adult steady state condition may not reveal a competitively disadvantaged at or before the CD44ϩ/ϪCD25ϩ TN potential defect in the progenitor cell population. To determine stage. Taken together, these data indicate that under competitive 2816 CHARACTERIZATION OF CCR9-DEFICIENT MICE

FIGURE 6. CCR9 expression in bone marrow and analysis of B cell development in CCR9Ϫ/Ϫ mice. A, CCR9 mRNA expression in bone marrow. Total RNA (20 ␮g) from bone marrow cells from B6 or Rag-1Ϫ/Ϫ mice was separated by gel electrophoresis, transferred to membranes, and hybridized with cDNA probes to mouse CCL25, mouse CCR9, or human EF-1␣. B, Immature bone marrow cells express CCR9. Bone marrow cells were stained with Abs directed to CD4, DX5, CD19, B220, and CCR9. Gated CD4ϩDX5ϪCD19ϪB220ϩ and CD4ϪDX5Ϫ CD19ϪB220ϩ cells were analyzed for surface CCR9 expression. C, Normal development of early B progenitor cells in CCR9Ϫ/Ϫ mice. Bone marrow cells from CCR9ϩ/ϩ or CCR9Ϫ/Ϫ mice were stained with mAbs directed to B220, CD43, DX5, CD19, and CD4. CD19ϪB220ϩ were gated and analyzed for CD4 and DX5 expression. The percentages of CD43ϩB220ϩ and CD19ϪB220ϩ cells in total bone marrow cells, and the percentages of CD4ϪDX5Ϫ Downloaded from and CD4ϪDX5Ϫ subsets among CD19ϪB220ϩ cells are indicated.

conditions, bone marrow cells from CCR9Ϫ/Ϫ mice are impaired DX5ϪCD19ϪB220ϩ bone marrow cells, which include early B http://www.jimmunol.org/ in their ability to reconstitute T cell, but not B cell, development in progenitors (37, 38), expressed low levels of CCR9 and could irradiated Rag-1Ϫ/Ϫ mice. respond to CCL25 (Fig. 6B and data not shown). In addition, CD4ϩDX5ϪCD19ϪB220ϩ cells were found to express high levels Discussion of CCR9 and could respond to CCL25. The lineage affiliation and ϩ Ϫ Ϫ ϩ In this study we examined the role of CCR9 in T and B cell de- differentiation potential of CD4 DX5 CD19 B220 cells remain velopment by generating CCR9-deficient mice by gene targeting. unclear, although phenotypically similar cells from bone marrow CCR9 is expressed on the surface of most ␣␤-lineage thymocytes that respond to CCL25 contain both B and T cell progenitors (36, ␥␦ ϩ and approximately half of all TCR thymocytes and T cells (20, 37, 39, 40). To investigate whether CCR9 is involved in regulating by guest on October 2, 2021 21). Mucosal T cells also express CCR9, and fetal blood prothy- the migration of T-progenitor cells to thymus, we performed com- mocytes and pre-pro-B cells migrate in response to CCL25 (11–15, petitive bone marrow transplantation experiments. The results Ϫ Ϫ 21, 23). Analysis of CCR9Ϫ/Ϫ mice revealed that 1) ␣␤-T cells demonstrated that CCR9 / bone marrow cells are competitively ϩ ϩ develop normally in adult CCR9Ϫ/Ϫ mice, but CCR9Ϫ/Ϫ bone disadvantaged compared with CCR9 / bone marrow cells in Ϫ Ϫ marrow cells exhibit a reduced capacity to repopulate the thymus their ability to repopulate the thymus of irradiated Rag-1 / mice of irradiated Rag-1Ϫ/Ϫ mice under competitive conditions com- (Fig. 7). These findings suggest three nonmutually exclusive pos- pared with CCR9ϩ/ϩ bone marrow cells; 2) CCR9Ϫ/Ϫ mice con- sibilities: 1) that CCR9 regulates the generation of prothymocytes tain increased numbers of peripheral ␥␦-T cells, but reduced num- in the bone marrow, 2) that CCR9 regulates the migration of pro- bers of small intestinal ␥␦TCRϩ IEL; and 3) B cell development thymocytes into the thymus or their migration or retention within is unaffected in CCR9Ϫ/Ϫ mice. the thymus, and 3) that CCR9 regulates the proliferation of early The finding that ␣␤-T cell development and thymocyte selection thymocytes in the thymus. Because immature TN thymocytes do appear unperturbed in adult CCR9Ϫ/Ϫ mice was unexpected given not express surface CCR9 and do not respond to CCL25 (21), it the fact that most thymocytes express high levels of CCR9 and appears unlikely that CCR9 is directly involved in the expansion of migrate to CCL25. The inability of CCR9Ϫ/Ϫ thymocytes to re- early thymocytes. Bleul et al. (12) reported that three chemokines spond to CCL25 demonstrates that these cells do not express an- (CCL25, CXCL12, and CCL21) are expressed in the thymic another receptor for CCL25. Several different chemokines are ex- lage, and both CCL25 and CXCL12 attract fetal blood prothymo- pressed in the thymus, and it is possible that they may share cytes. Similar to CCR9Ϫ/Ϫ mice, mice deficient in CXCL12 or its overlapping targets and therefore compensate for the loss of receptor, CXCR4, also showed no obvious abnormality in T cell CCR9. Histological examination of the thymus revealed no obvi- development (41–43). On the basis of these findings, we speculate ous abnormalities in CCR9Ϫ/Ϫ mice; however, more detailed lo- that CCR9 in addition to other chemokine receptors such as calization studies of specific thymocyte subsets may be required to CXCR4 may play an important and partially redundant role in detect subtle intrathymic migration defects. regulating the migration of prothymocytes into the thymus. Our Previous results indicate that prothymocytes in fetal blood re- inability to detect surface expression of CCR9 on immature TN spond to CCL25 (12). This observation together with the finding thymocyte subsets is not necessarily in conflict with this idea, as that CCL25 is not expressed in bone marrow (Fig. 6A) suggested the population of CCR9ϩ prothymocytes may be extremely small, that CCR9 may participate in the migration of T-progenitor cells or CCR9 may be down-regulated when prothymocytes enter the from bone marrow to thymus. Although we were unable to detect thymus. CCR9 surface expression on immature TN thymocytes from adult ␥␦-T cell development and/or homeostasis were clearly altered mice, and these cells did not respond to CCL25, CCR9ϩ cells in CCR9Ϫ/Ϫ mice. Although CCR9Ϫ/Ϫ mice contained normal were present in adult bone marrow (Fig. 6, A and B). CD4Ϫ numbers of ␥␦TCRϩ thymocytes, the number of ␥␦-T cells was The Journal of Immunology 2817 Downloaded from http://www.jimmunol.org/

FIGURE 7. Competitive bone marrow transplantation into irradiated RagϪ/Ϫ mice. Bone marrow cells were isolated from femurs of CCR9ϩ/ϩ (CD45.2), CCR9Ϫ/Ϫ (CD45.2), and B6.SJL-Ptprca/BoAiTac mice (B6.CD45.1; Taconic Farms). A total of 2 ϫ 106 cells consisting of mixtures of CCR9ϩ/ϩ or CCR9Ϫ/Ϫ bone marrow with B6.CD45.1 bone marrow was injected i.v. into lethally irradiated (9.5 Gy) Rag-1Ϫ/Ϫ B6 mice (CD45.2). One to 2 mo after transplantation, thymocytes and lymph node B cells were examined for the expression of CD45.2 by FACS. A, Representative FACS plots showing the expression of CD4, CD8␣, and CD45.2 on thymocytes from irradiated Rag-1Ϫ/Ϫ mice injected with equal numbers of bone marrow cells from CCR9ϩ/ϩ and B6.CD45.1 mice (upper left panel) or CCR9Ϫ/Ϫ and B6.CD45.1 mice (upper right panel). Histograms show the expression of CD45.2 on total Ϫ Ϫ Ϫ thymocytes. B, Representative FACS plots showing the expression of CD44 and CD25 on gated (CD4 CD8 CD3 (TN)) thymocytes from irradiated by guest on October 2, 2021 Rag-1Ϫ/Ϫ mice injected with equal numbers of bone marrow cells from CCR9ϩ/ϩ and B6.CD45.1 mice (upper left panel) or CCR9Ϫ/Ϫ and B6.CD45.1 mice (upper right panel). Histograms show CD45.2 expression on CD44ϩ/ϪCD25ϩ or CD44ϪCD25Ϫ thymocytes. C, Plot of the percentage of CD45.2ϩ cells in total thymocytes from irradiated RagϪ/Ϫ mice injected with the indicated ratios of bone marrow cells. The percentage of thymocytes derived from CCR9Ϫ/Ϫ bone marrow cells was signiﬁcantly lower than that of CCR9ϩ/ϩ bone marrow-derived thymocytes (p Ͻ 0.01). D, Plot of the percentage of CD45.2ϩ cells in lymph node B cells (B220ϩ) from irradiated Rag-1Ϫ/Ϫ mice injected with the indicated ratios of bone marrow cells. E, Data from mice injected with mixtures of bone marrow from CCR9ϩ/ϩ (CD45.2) and B6.CD45.1 mice; F, data from mice injected with mixtures of bone marrow from CCR9Ϫ/Ϫ (CD45.2) and B6.CD45.1 mice.

increased in secondary lymphoid organs (spleen and lymph nodes; found that the percentage of ␥␦TCRϩ IEL in the large intestine Fig. 4A). All TCR-V␥/V␦ pairs examined were increased in lymph was increased (Fig. 5, B and C). The large intestinal ␥␦TCRϩ IEL nodes and spleen of CCR9Ϫ/Ϫ mice (Fig. 4B and data not shown). in CCR9Ϫ/Ϫ mice were phenotypically similar to those in In addition, we could not detect any significant difference in the CCR9ϩ/ϩ mice (i.e., they did not resemble small intestinal IEL; expression of CD44, CD45RB, and other surface markers (e.g., data not shown), indicating that the increase in large intestinal ␣ ␥␦ Ϫ/Ϫ ␥␦ ϩ CD62L and IEL integrin) on peripheral -T cells from CCR9 TCR IEL is not due to migration of cells from the small in- and CCR9ϩ/ϩ mice (data not shown), suggesting that the increased testine to the large intestine. number of peripheral ␥␦-T cells in CCR9Ϫ/Ϫ mice is not due to the All IEL from human small intestine express CCR9 and respond accumulation of one particular subpopulation of ␥␦-T cells. The to CCL25 (13–15), and in mice both ␥␦TCRϩ and ␣␤TCRϩ IEL kinetics of thymocyte development are much more rapid for ␥␦- have been shown to express CCR9 mRNA (21). Thus, CCR9 than ␣␤-T cells, and ␥␦-T cells appear to be dependent on the might be important for the recruitment of mature ␥␦-T cells to the thymic environment for a relatively brief period during their de- intestinal mucosa. Indeed, the observation that large intestinal IEL velopment (44). Thus, in the absence of CCR9, ␥␦-T cells may be were not decreased in CCR9Ϫ/Ϫ mice localizes the defect to the generated in higher numbers and immigrate more rapidly into the site of CCL25 production (Fig. 5, A and B). Consistent with this periphery. idea is the finding that small intestinal ␣␤TCRϩCD8␣␤ϩ IEL, In contrast to lymph nodes and spleen, we observed that the which are thought to be thymically derived and would therefore percentage of small intestinal ␥␦TCRϩ IEL was consistently de- migrate from the periphery to the small intestine, are also reduced creased in CCR9Ϫ/Ϫ mice. In the gastrointestinal tract, CCL25 in CCR9Ϫ/Ϫ mice (Fig. 5F). Another possibility is that in the ab- expression is restricted to the small intestinal epithelium (Fig. 5A) sence of CCR9, ␥␦TCRϩ IEL fail to be retained in the small in- (11, 13–15). Interestingly, although the percentage of ␥␦TCRϩ testine. However, the preferential loss of TCR-V␥5/␦4ϩ IEL and IEL in the small intestine of CCR9Ϫ/Ϫ mice was decreased, we our inability to find these cells in lymph node or spleen (Fig. 5E 2818 CHARACTERIZATION OF CCR9-DEFICIENT MICE and data not shown) suggest that there may be a direct role for 10. Wilkinson, B., J. J. T. Owen, and E. J. Jenkinson. 1999. Factors regulating stem CCR9 in V␥5/␦4ϩ IEL development or that these cells may fail to cell recruitment to the fetal thymus. J. Immunol. 162:3875. 11. Wurbel, M.-A., J.-M. Philippe, C. Nguyen, G. Victorero, T. Freeman, survive if they are unable to home to the proper site. The accu- P. Wooding, A. Miazek, M.-G., Mattei, M. Malissen, B. R. Jordan, et al. 2000. mulation of diverse subsets of ␥␦-T cells in the periphery of The chemokine TECK is expressed by thymic and intestinal epithelial cells and Ϫ/Ϫ attracts double- and single-positive thymocytes expressing the TECK receptor CCR9 mice could also reflect the pool of cells that initially CCR9. Eur. J. Immunol. 30:262. migrate to the intestine and may differ significantly from the pop- 12. Bleul, C. C., and T. Boehm. 2000. Chemokines define distinct microenviron- ulation that ultimately becomes established as IEL. Finally, the ments in the developing thymus. Eur. J. Immunol. 30:3371. ␥␦ 13. Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, increase in peripheral -T cells may reflect a shift in a dynamic E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al. 1999. Human G protein-coupled equilibrium between the small intestine and the peripheral pool. receptor GPR-9–6/CC chemokine receptor 9 is selectively expressed on intesti- The lower numbers of ␥␦TCRϩ IEL in CCR9Ϫ/Ϫ mice even in the nal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis. J. Exp. Med. 190: face of what may be a compensatory increase in these cells in the 1241. periphery suggests an important role for CCR9 in establishing this 14. Kunkel, E. J., J. J. Campbell, G. Haraldsen, J. Pan, J. Boisvert, A. J. Roberts, equilibrium. E. C. Ebert, M. A. Vierra, S. B. Goodman, M. C. Genovese, et al. 2000. Lym- phocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine CCR9 and CCL25 mRNA are also detected in the small intes- (TECK) expression distinguish the small intestinal immune compartment: epi- Ϫ Ϫ tine of Rag-1 / mice, which lack mature lymphocytes (21), rais- thelial expression of tissue-specific chemokines as an organizing principal in ing the possibility that CCR9 plays a role in early mucosal T cell regional immunity. J. Exp. Med. 192:761. 15. Papadakis, K. A., J. Prehn, V. Nelson, L. Cheng, S. W. Binder, P. D. Ponath, development and/or recruitment of IEL precursors to the small D. P. Andrew, and S. R. Targan. 2000. The role of thymus-expressed chemokine intestine. Cryptopatches are multiple clusters of c-kitϩIL- and its receptor CCR9 on lymphocytes in the regional specialization of the mu- 7RϩThy1ϩ lymphocytes located in the crypt lamina propria of the cosal immune system. J. Immunol. 165:5069. 16. Zaballos, A., J. Gutie´rrez, R. Varona, C. Ardavıń, and G. Ma´rquez. 1999. Iden- Downloaded from murine intestine (45). CCR9 may be involved in cryptopatch for- tification of the orphan chemokine receptor GPR-9–6 as CCR9, the receptor for mation and/or extrathymic ␥␦TCRϩ IEL development, perhaps by the chemokine TECK. J. Immunol. 162:5671. regulating the migration of progenitor cells from the fetal liver, 17. Youn, B.-S., C. H. Kim, F. O. Smith, and H. E. Broxmeyer. 1999. TECK, an ϩ efficacious chemoattractant for human thymocytes, uses GPR-9–6/CCR9 as a fetal thymus, or adult bone marrow to the small intestine. ␥␦TCR specific receptor. Blood 94:2533. IEL are greatly reduced in nude mice and neonatal thymectomized 18. Yu, C.-R., K. W. C. Peden, M. B. Zaitseva, H. Golding, and J. M. Farber. 2000. ␤ mice (46). Transplantation of fetal or neonatal thymus, but not CCR9A and CCR9B: two receptors for the chemokine CCL25/TECK/Ck -15

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