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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 de- velopment (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 Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 2812 CHARACTERIZATION OF CCR9-DEFICIENT MICE 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).