Immune System the Regional Specialization of the Mucosal And

The Role of Thymus-Expressed Chemokine and Its Receptor CCR9 on Lymphocytes in the Regional Specialization of the Mucosal Immune System This information is current as of September 25, 2021. Konstantinos A. Papadakis, John Prehn, Viera Nelson, Lorna Cheng, Scott W. Binder, Paul D. Ponath, David P. Andrew and Stephan R. Targan J Immunol 2000; 165:5069-5076; ; doi: 10.4049/jimmunol.165.9.5069 Downloaded from http://www.jimmunol.org/content/165/9/5069

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Role of Thymus-Expressed Chemokine and Its Receptor CCR9 on Lymphocytes in the Regional Specialization of the Mucosal Immune System1

Konstantinos A. Papadakis,2* John Prehn,* Viera Nelson,† Lorna Cheng,† Scott W. Binder,† Paul D. Ponath,‡ David P. Andrew,‡ and Stephan R. Targan*

Chemokines play an important role in the migration of leukocytes at sites of inﬂammation, and some constitutively expressed chemokines may direct lymphocyte trafﬁcking within lymphoid organs and peripheral tissues. Thymus-expressed chemokine (TECK or Ck␤-15/CCL25), which signals through the chemokine receptor CCR9, is constitutively expressed in the thymus and ␣ ␤ small intestine but not colon, and chemoattracts a small fraction of PBLs that coexpress the integrin 4 7. Here we show that

TECK is expressed in the human small bowel but not colon by endothelial cells and a subset of cells in intestinal crypts and lamina Downloaded from propria. CCR9 is expressed in the majority of freshly isolated small bowel lamina propria mononuclear cells (LPMC) and at signiﬁcantly higher levels compared with colonic LPMC or PBL. TECK was selectively chemotactic for small bowel but not colonic LPMC in vitro. The TECK-induced chemotaxis was sensitive to pertussis toxin and partially inhibited by Abs to CCR9. TECK attracts predominantly the T cell fraction of small bowel LPMC, whereas sorted CD3؉CCR9؉ and CD3؉CCR9؊ lymphocytes produce similar Th1 or Th2 cytokines at the single cell level. Collectively, our data suggest that the selective expression of TECK ؉ in the small bowel underlie the homing of CCR9 intestinal memory T cells to the small bowel rather than to the colon. This http://www.jimmunol.org/ regional specialization implies a segregation of small intestinal from colonic immune responses. The Journal of Immunology, 2000, 165: 5069–5076.

hemokines (chemotactic cytokines) are small, 6- to 14- chemokine 1, and CCR9, which binds thymus-expressed chemokine kDa heparin-binding proteins, which play a role in a variety (TECK) only, among others (5–15). Chemokines were identified by C of biological processes, most notably leukocyte chemotaxis their ability to direct extravasation of inflammatory cells during in- (1–3). They are classified as C, CC, CXC, and CX3C based on the fection (1, 2). However, recent data identified several chemokines that positioning of cysteine residues that form two disulfide bonds (3). are expressed constitutively in lymphoid and extra-lymphoid tissues, by guest on September 25, 2021 Chemokines mediate their effects through G protein-coupled seven indicating that these chemokines might have homeostatic function by transmembrane domain receptors, which are currently divided into regulating lymphocyte trafficking to or within lymphoid organs and in four families based on the type of chemokine that they bind; they are peripheral tissues (3, 16–21). Certain chemokines, such as stromal CXCR1 to CXCR5, and CCR1 to CCR9, XCR1, or CX3CR1 (3). cell-derived factor 1 (SDF-1), 6-C-kine, and MIP-3␤ can also stim- Most chemokines recognize several receptors, and a single receptor ulate leukocyte adhesion and arrest on endothelium by triggering in- can bind more than one chemokine (4). However, some receptors bind tegrin activation (22, 23). It is thought that the combined expression a single chemokine such as CCR6, which binds macrophage inflam- of adhesion molecules and chemokine receptors on the cell surface 3 ␣ matory protein (MIP) -3 , CXCR5, which binds B cell-attracting provide an “address code” for leukocyte migration to different sites (24). In contrast to naive T cells, memory/effector cells migrate *Department of Medicine, Division of Gastroenterology and Inflammatory Bowel mostly through peripheral tissues, and this process is controlled by Disease Center, Cedars-Sinai Medical Center, University of California, Los Angeles School of Medicine, Los Angeles, CA 90048; †Department of Pathology and Labo- the expression of different sets of integrins and chemokine recep- ratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA 90048; and ‡Leu- tors (25–28). Thus, naive T cells express CXCR4, the receptor for koSite, Cambridge, MA 02142 SDF-1, and CCR7, the receptor for EBV-induced molecule 1 li- 1 This work was supported by National Institutes of Health Grants DK-46763 and DK-56328. gand chemokine and secondary lymphoid tissue chemokine (also Received for publication May 22, 2000. Accepted for publication July 31, 2000. called 6-C-kine). Mice deficient in CCR7 or secondary lymphoid tissue chemokine have defective homing of naive T cells to sec- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ondary lymphoid organs (16, 29). Gene knockout studies have es- with 18 U.S.C. Section 1734 solely to indicate this fact. tablished that CXCR5 is required for B cell migration to B cell 2 Address correspondence and reprint requests to Dr. Konstantinos A. Papadakis, follicles of spleen and Peyer’s patches (PP; Ref. 30). In addition, Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, D-4062, Los Angeles, CA 90048. E-mail address: [email protected] the defects in lymph node (LN) development observed in lympho- or Dr. Stephan R. Targan, Inflammatory Bowel Disease Center, Cedars-Sinai Medical toxin-␣ and TNF knockout mice have been attributed, at least in Center, 8700 Beverly Boulevard, D-4063, Los Angeles, CA 90048. E-mail address: part, to decreased production of chemokines by stromal LN cells [email protected] (31). Collectively, these data suggest an important role of certain 3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; LN, lymph node; LPMC, lamina propria mononuclear cells; PP, Peyer’s patches; TECK, chemokines in regulating the homing of specific T cell subsets and thymus-expressed chemokine; hTECK, human TECK; IECs, intestinal epithelial other immune cells into microanatomic compartments of second- cells; MAdCAM-1, mucosal addressin cell-adhesion molecule; mTECK, murine TECK; IELs, intraepithelial lymphocytes; SDF-1, stromal cell-derived factor 1; MLN, ary lymphoid organs. Certain chemokine receptors are also pref- mesenteric lymph node; MFI, mean fluorescence intensity. erentially expressed on naive T cells under Th1 or Th2 polarizing

conditions in vitro. Th1 cells predominantly express CXCR3 and After washing twice, cells were resuspended in 400 ␮l of 1% paraformal- CCR5 (32–36). In contrast, Th2 cells express CCR3, CCR4, and dehyde in PBS and analyzed by FACS (Becton Dickinson, Mountain View, 4 CCR8 (3, 24, 32, 33, 35, 36). Recently, the orphan chemokine CA). Events (10 ) were routinely collected and analyzed using Lysis II software (Becton Dickinson Immunocytometry Systems, San Jose, CA). receptor GPR-9-6, now designated CCR9, was found to be ex- Both the percentage of positive cells and the mean ﬂuorescence intensity pressed on a small percentage (2–4%) of circulating memory T (MFI) of the cells were determined. ␣ ␤ cells, all of which express the mucosal homing ligand 4 7 (9). The ligand for CCR9, TECK, also CCL25/Ck␤-15 according to RT-PCR the recent chemokine/chemokine receptor nomenclature (3), is se- Total RNA was extracted from small bowel or colonic intestinal mucosa lectively expressed in the thymus and small intestine (9, 13, 37, and freshly isolated small bowel or colonic IECs and LPMC using the 38). In addition, CCR9 mRNA is expressed in the thymus, small RNeasy Kit as recommended by the manufacturer (Qiagen, Valencia, CA). One microgram of RNA was reverse-transcribed into cDNA with oligo(dT) intestine, and at lower levels in the spleen, suggesting that the in a 20-␮l volume using a Thermoscript RT-PCR System (Life Technol- CC-chemokine TECK and its receptor, CCR9, may play an im- ogies, Grand Island, NY) according to a standard protocol. Primers were portant role in T cell maturation and in the intestinal immune re- designed as described elsewhere (9). Primers for TECK were: sense 5Ј- sponse (9). TCGAAGAAGCTTATGAACCTGTGGCTCCTG-3Ј antisense 5Ј-AA Ј The potential importance of CCR9 and TECK in mucosal im- GAAGTCTAGATCACAGTCCTGAATTAGC-3 (product 453 bp). Two microliters of the reaction cDNA was mixed with 10 mM dNTP, 10 ␮M munity prompted us to study the expression of this chemokine/ ␮ primers, 50 mM MgCl2,and5U/ l of Platinum Taq DNA polymerase in chemokine receptor pair in the normal small and large bowel. Our a 50-␮l volume as recommended by the manufacturer (Life Technologies). data suggest that the TECK/CCR9 chemokine/chemokine receptor The cycle parameters were an initial melt at 95°C for 2 min, then 35 cycles:

95°C, 30 s; 55°C, 30 s; and 72°C, 1 min, followed by a ﬁnal extension of Downloaded from pair is important for the regional specialization of intestinal im- Ј ␣ ␤ 72°C, 7 min. Ampliﬁcation with G3PDH primers (sense 5 -TGAAG munity and the combined expression of CCR9/ 4 7 on the cell GTCGGAGTCAACGGATTTGGT-3Ј, antisense 5Ј-CATGTGGGCCAT surface may provide a small intestinal “address code” for circu- GAGGTCCACCAC-3Ј) (product 983 bp) (Clontech, Palo Alto, CA) was lating intestinal memory T cells. examined in identical conditions as an internal control to demonstrate equivalence of template. The PCR products were visualized with ethidium Materials and Methods bromide after 1.5% agarose gel electrophoresis.

Purification of lamina propria mononuclear cells (LPMC), Cytokine detection at the single cell level http://www.jimmunol.org/ PBMC, mesenteric lymph node (MLN) lymphocytes, and Freshly isolated LPMC or sorted mucosal T cells were stimulated with intestinal epithelial cells (IECs) 10Ϫ7 M PMA and 1 ␮g/ml ionomycin for 4 h. Brefeldin A (10 ␮g/ml) was Intestinal specimens were obtained from patients undergoing surgical re- added to the culture after2hofstimulation to block cytokine secretion. 6 section of the colon (with colon carcinoma) or small intestine usually dur- Unseparated cells (10 cells) were surface stained and washed twice as ing the second stage of a prior ileal anal-pouch anastomosis at Cedars-Sinai mentioned above and subsequently fixed with 4% paraformaldehyde and Medical Center (Los Angeles, CA). Approval for the use of human subjects permeabilized with saponin. Fixed and permeabilized cells were stained ␥ was obtained from the Institutional Review Board at Cedars-Sinai Medical with FITC anti-IFN- and PE anti-IL-4 mAbs (PharMingen) and analyzed Center. In this study, all tissue specimens were taken from an uninvolved by FACS (Becton Dickinson). area of resected colon or small bowel. LPMC were isolated using a tech- nique modified from that described previously (39). PBMC were isolated Chemotaxis assay by guest on September 25, 2021 from normal healthy volunteers by separation on Ficoll-Hypaque gradients. Cell migration was evaluated using a 48-well chemotaxis chamber (Neu- The cells were subsequently washed three times with HBSS and resus- roprobe, Cabin John, MD). TECK, diluted in HEPES-buffered RPMI 1640 pended in culture media (RPMI 1640 with 10% FCS) at a concentration of supplemented with 1% BSA, was added to the lower wells, and 105 cells ϫ 6 2 10 /ml. MLN lymphocytes were isolated following mechanical dis- in the same buffer to the upper wells. Polyvinylpyrrolidone-free polycar- ruption of LNs. IECs from small bowel or colon were isolated as previ- bonate membranes (Neuroprobe) with 3-␮m pores were used. After incu- ously described (40). bation for 120 min at 37°C, the cells that had migrated through the pores ␮ Abs and reagents to the lower wells were counted by FACS. A known number of 3.2- m fluorescent microsphere beads (PharMingen) was added to each sample Anti-CD3, -CD4, -CD8, -CD19, -CD56, -HLA-DR, -CD45RO dye-linked before analysis to determine the absolute number of migrating cells. The mAbs for immunofluorescence studies were obtained from Caltag (South assay was performed in triplicate. San Francisco, CA). Anti-CD25, -CD69, and -CD95 were obtained from PharMingen (San Diego, CA). TECK and SDF-1␣ were purchased from Immunohistochemistry PeproTech (Rocky Hill, NJ). Sections were fixed and stained as previously described (41). Briefly, fixed ␮ Generation of anti-TECK and anti-CCR9 mAbs sections (6 m) from normal small bowel and colonic mucosa, or thymus, were deparaffinized and treated with citrate buffer (pH 6). The sections The mAb to human TECK (hTECK; clone LS202 5A9, IgG1) was gener- were then incubated with anti-hTECK Ab (clone LS202 5A9, IgG1) fol- ated by i.p immunization of BALB/c mice at 3-wk intervals with 10 ␮gof lowed by a goat anti-mouse Ab (Dako, Carpinteria, CA) at a 1:20 dilution. TECK (Peprotech) in CFA, IFA, and finally PBS. Fusions were performed The sections were subsequently incubated with a mouse peroxidase anti- after at least four immunizations 3 days after the last boost by fusion with peroxidase (Dako) at 1:100 dilution. Following wash, the sections were SP2/0 myeloma cells (American Type Culture Collection, Manassas, VA). developed with DAB substrate-chromogen (Dako). Sections were counter- Fusions were screened by ELISA with plates coated with hTECK, and the stained with hematoxylin (Fisher Scientific, Pittsburgh, PA). positive hybridomas were subcloned. Of 20 anti-TECK mAbs tested only two, designated 4G1 and 5A9, were found to stain frozen sections of thy- Statistical analysis mus. The mAb to human CCR9 has been previously described (9). Where indicated Student’s t test was used to calculate statistical signifi- Flow cytometry cance for difference in a particular measurement between different groups. Values of p Ͻ 0.05 were considered statistically significant. Freshly isolated or cultured LPMC or PBMC (2.5–5.0 ϫ 105) were washed twice with 1 ml of PBS supplemented with 0.1% BSA and 0.1% azide. The Results cells were resuspended in 100 ␮l of 10% human Ab serum to block non- specific Fc binding for 15 min. For the staining of surface Ags, cells were CCR9 is highly expressed on small bowel compared with incubated with the mAb 3C3 for 30 min on ice, washed with PBS/BSA/ colonic or peripheral blood T lymphocytes azide, and incubated with a secondary goat anti-mouse anti-Fab (HϩL) ϩ tricolor or goat anti-mouse IgG2b-PE for 30 min on ice. The cells were We measured the percentage CCR9 LPMC in the small bowel washed again with PBS/BSA/azide and incubated with mouse IgG for 15 and colon by flow cytometry using a CCR9-specific mAb, desig- min. FITC- and PE-conjugated mAb for surface Ag were used for 30 min. nated 3C3. In cross-reactivity studies, 3C3 did not cross-react with The Journal of Immunology 5071

CCR1-7 or CXCR1-4 transfectants (9). Three color immunofluo- Table I. MFI of CCR9 expression in small bowel, colonic, and ϩ ϩ ϩ ϩ rescence analysis of LPMC from small bowel or colon and PBMC peripheral blood CD3 CD4 and CD3 CD8 lymphocytes showed that CCR9 is expressed predominantly in small bowel compared with colonic or peripheral blood CD3ϩ T lymphocytes. CD3ϩCD4ϩ CD3ϩCD8ϩ a b CCR9 was expressed at high levels in both the CD8 and CD4 Cell Source Mean (range) Mean (range) ϩ subsets of small bowel CD3 cells. As shown in Fig. 1, CCR9 was Small bowel LPMC (n ϭ 9) 81 (45–133) 76.3 (44–127) expressed in 67% (range, 57–76%) of CD3ϩCD4ϩ small bowel Colonic LPMC (n ϭ 9) 37 (24–60) 33 (19–54) ϭ compared with 20% (range, 15–25%) of colonic ( p Ͻ 0.0001) and PBL (n 9) 32 (20–42) 23.4 (15–30) ϩ ϩ 4% (range, 3–5%) of peripheral blood CD3 CD4 lymphocytes. a Small bowel vs colonic LPMC, p ϭ 0.01; small bowel LPMC vs PBL, p Ͻ The differences between groups were statistically significant ( p Ͻ 0.002; and colonic LPMC vs PBL, p ϭ 0.2. b Small bowel vs colonic LPMC, p Ͻ 0.0001; small bowel LPMC vs PBL, p Ͻ 0.0001). Similar differences in CCR9 expression were observed 0.0001, and colonic LPMC vs PBL, p ϭ 0.03. between small bowel, colonic, or peripheral blood CD3ϩCD8ϩ lymphocytes. CCR9 was expressed in 58% (range, 47–70%) compared with 10% (range, 6–14%) and 2% (range, 1.4–2.6%) of expressed by thymic dendritic cells (37) but two recent reports small bowel, colonic, or peripheral blood CD3ϩCD8ϩ cells, re- showed it to be expressed by thymic and small IECs (13, 38). spectively (Fig. 1). In addition, the density of CCR9 expression, as Therefore, we studied the expression of TECK in human intestinal assessed by the MFI in each lymphocyte subset showed that CCR9 tissues. First we analyzed TECK expression by RT-PCR in whole is expressed at higher levels in small bowel compared with colonic small bowel and colonic mucosa, as well as in IECs and LPMC or peripheral blood T cells in both the CD4 and CD8 compartment isolated from small bowel and colon. Fig. 4 shows that TECK was Downloaded from (Table I). We further analyzed the expression of CCR9 by flow detected by RT-PCR in whole small bowel mucosa, as well as in cytometry on freshly isolated MLN lymphocytes draining small small bowel IECs and LPMC. TECK message was absent from bowel and colon. CD3ϩCCR9ϩ lymphocytes were significantly whole colonic mucosa and colonic IECs or LPMC. To investigate enriched in small bowel vs colonic MLN (mean, 69 vs 11%, n ϭ more precisely the distribution of TECK expression in small 3, p Ͻ 0.05). A representative FACS analysis of CCR9 expression bowel, we performed immunohistochemistry using a mAb, desig-

in MLN lymphocytes is shown in Fig. 2. Collectively, our data nated 5A9, against hTECK. Thymus tissue was used as positive http://www.jimmunol.org/ show that the chemokine receptor CCR9 is expressed in a much control for hTECK staining. As shown in Fig. 5, TECK staining larger percentage and at a higher density in small bowel compared was detected in the small bowel and thymus but not colon (com- with colonic lamina propria and draining MLN T lymphocytes. pare a–c, and f with d). Strong TECK immunoreactivity was de- We next analyzed the phenotype of CCR9-expressing small tected in a subset of cells with elongated processes in the small bowel LPMC. As shown in Fig. 3, CCR9 was expressed predom- bowel intestinal crypts and the lamina propria predominantly in inantly on CD3ϩ small bowel LPMC, although a signiﬁcant per- areas of lymphocyte aggregates (Fig. 5b). TECK staining was not centage of CD3Ϫ cells also express CCR9. Also CCR9 was ex- detected in mucosal lymphocytes or surface epithelial cells. pressed on a subset of B and NK cells. Almost all CCR9ϩ LPMC, TECK-immunoreactive cells were also detected in small bowel but whether from small bowel or colon, coexpressed CD45RO, CD69, not colonic endothelial cells (compare Fig. 5, a inset and c with d). by guest on September 25, 2021 CD95, and a signiﬁcant percentage were also HLA-DRϩ (Fig. 3, In thymic sections, TECK immunoreactivity was observed in the stro- and data not shown). This is consistent with the known phenotype mal component of the thymic medulla and cortex, as has been shown of mucosal lymphocytes as highly activated memory cells (39). previously for TECK expression in murine thymus (Fig. 5f).

TECK is selectively expressed in the small bowel but not colon TECK mediates chemotaxis of small bowel but not colonic It has been shown that hTECK message was detected by RT-PCR or PBLs and Northern blot in the thymus and small bowel, but not colon (9, TECK has been shown to induce Ca2ϩ mobilization and chemo- 37). In mice, murine TECK (mTECK) was initially reported to be taxis of CCR9 transfectants (9, 10, 14), suggesting that CCR9 is a

FIGURE 1. CCR9 expression in peripheral blood, colonic, and small bowel T lymphocytes. Freshly isolated mononuclear cells from normal small bowel or colonic mucosa and PBL were stained with 3C3, anti-CD3, -CD4, or -CD8 mAb and analyzed by FACS. The cells were gated on CD3ϩ lymphocytes. The data represent a total of nine independent experiments from different donors with the mean Ϯ SEM shown. Differences between all groups were statistically signiﬁcant (p Ͻ 0.0001). 5072 CCR9-POSITIVE T CELLS/TECK EXPRESSION IN HUMAN SMALL BOWEL IMMUNITY

TECK may be involved in the selective trafficking of CCR9-expressing lymphocytes to the small bowel instead of colon. There- fore, we examined whether small bowel and colonic LPMC respond differentially to TECK in a chemotaxis assay. In bulk migration experiments, TECK chemoattracts freshly isolated small bowel but not colonic LPMC or PBMC (Fig. 6a). The migrating small bowel LPMC when plotted vs increasing concentration of TECK revealed a bell-shaped curve typically observed with other FIGURE 2. CCR9 expression in MLNs lymphocytes draining small chemokines. The optimal chemotactic concentration of TECK was bowel or colon. Lymphocytes were stained with 3C3 or isotype control Ab, 500 nM for small bowel lymphocytes, which is within the range followed by PE-conjugated anti-IgG2b and analyzed by flow cytometry. (1–1000 nM) of chemotactic activity seen with other chemokines Representative one of three independent experiments from different donors is shown. (9) (Fig. 6a). Checkerboard analysis established that TECK induced chemotaxis and not chemokinesis of small bowel LPMC (data not shown). The inability of colonic LPMC to migrate in specific chemotactic receptor for TECK. In addition, the small per- response to TECK was not due to a general migratory defect of centage of PBMC that respond by chemotaxis to TECK express those cells compared with small bowel LPMC because both mi- ␣ ␤ ␣ high levels of the mucosal homing ligand 4 7 (9). In addition, the grated to SDF-1 , which signals through CXCR4 (23) (data not

selective expression of the chemokine TECK in the small bowel shown). Migration in response to TECK could be partially blocked Downloaded from and its receptor CCR9 in small bowel lymphocytes suggest that by preincubation of lymphocytes with anti-CCR9, but not by anti- http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. CCR9 expression on various popula- tions of small bowel lamina propria lymphocytes. a, Two-color staining of CCR9 (vertical axis)onT (CD3ϩ), B (CD19ϩ), and NK cells (CD56ϩ), re- spectively (horizontal axis). b, CCR9 expression (vertical axis)onCD3ϩ-gated small bowel lymphocytes, with subset or activation-related markers (horizontal axis), analyzed by three-color immuno- ﬂuorescence. Quadrants were set according to the staining of control mAbs. A representative of two experiments is shown. The Journal of Immunology 5073

non-T/non-B cells (CD3Ϫ CD19Ϫ) cells and CD4ϩ or CD8ϩ subsets of T cells. LPMC that migrated to the lower wells of the chemotaxis chamber were pooled and stained with mAb for CD3, CD4, CD8, CD45, and CD19. The percentage of cells of each phenotype that migrated to TECK was analyzed by FACS. As shown in Fig. 7, TECK was primarily chemotactic for T cells, both the CD4ϩ and CD8ϩ subsets. The percentage of T cells was 85% (60% CD4ϩ, 20% CD8ϩ) in the migrating population compared with 56% (39% CD4ϩ, 13% CD8ϩ) in the starting population. TECK was not chemotactic for B lymphocytes and non-T/non-B mononuclear cells (Fig. 7). FIGURE 4. Expression of TECK in small bowel and colon. RT-PCR of RNA isolated from whole intestinal mucosa (1), IECs (2), or LPMC (3) CCR9 expression in small bowel lymphocytes does not correlate from colon and small bowel. RTϪ shows the PCR product of small bowel LPMC RNA performed as in lane 3 except that the RT reaction was with the production of Th1 or Th2 cytokines omitted. Distinct profiles of chemokine receptors are acquired during in vitro differentiation of naive T lymphocytes into Th1 or Th2 subsets. For example, Th1 cells preferentially express CXCR3 and CCR3 mAb (Fig. 6b). Preincubation of the cells with pertussis CCR5 (32–36), and Th2 cells express CCR3, CCR4, and CCR8 toxin completely inhibited the migration in response to TECK, (24, 32, 33, 35, 36). Therefore, we examined whether CCR9 ex- Downloaded from ␣ consistent with G i protein-coupled signaling through CCR9 (9), pression in mucosal T cells defines a Th1 or Th2 cytokine-pro- as has been shown for other chemokine receptors (1–3). ducing phenotype. We examined cytokine production at the single cell level by intracellular cytokine staining. In initial experiments, Small bowel LPMC migrating to TECK is primarily T we found no differences in the percentage of IFN-␥-producing lymphocytes (Th1) or IL-4-producing (Th2) cells between small bowel and co- To further characterize the mononuclear cell subsets that respond lonic CD3ϩ lymphocytes despite a significant difference in the http://www.jimmunol.org/ to TECK, we analyzed the phenotype of small bowel LPMC that expression of CCR9 between these lymphocyte subsets (data not migrated to optimal concentrations (500 nM) of TECK. We used shown). To further confirm the cytokine profile of CCR9ϩ muco- flow cytometry to identify T cells (CD3ϩ), B cells (CD19ϩ), or sal T cells, CD3ϩ cells from the small bowel were sorted into by guest on September 25, 2021

FIGURE 5. Analysis of TECK protein expression in small bowel and colonic intestinal mucosa and thymus. a, TECK immunohistochemistry reveals scattered cells in intestinal crypts and endothelial cells of the small intestine (arrows). IECs in the villi are nonreactive. Magnification, ϫ40. b, TECK immunoreactive cells are present in intestinal crypts and lamina propria from small bowel. Mag- nification, ϫ400. c, Immunohistochemistry for TECK reveals staining of small bowel endothelium. Magnification, ϫ400. d, TECK immunohistochemistry reveals no staining of colonic crypts or endothelium. Magnification, ϫ40. e, Negative control. Small bowel mucosa does not stain with an irrele- vant isotype control Ab. Magnification, ϫ40. f, Pos- itive control. Staining of thymus tissue reveals scattered TECK-immunoreactive cells in the cortex and medulla. Magnification, ϫ400. 5074 CCR9-POSITIVE T CELLS/TECK EXPRESSION IN HUMAN SMALL BOWEL IMMUNITY

ﬁle and that CCR9 expression is rather linked to a phenotype with selective homing potential to the small bowel mucosa.

Discussion Ag-reactive memory and effector cells induced in response to intestinal immunization traffic preferentially into the intestinal wall and/or into the intestine-associated lymphoid organs (28, 42). Mu- cosal addressin cell-adhesion molecule (MAdCAM-1) is expressed on high-endothelial venules of PP, MLN, and postcapillary venules in the intestinal lamina propria and interact with the leu- ␣ ␤ ␣ ␤ kocyte integrin 4 7 (43). MAdCAM-1/ 4 7 interaction seems to ␣ ␤ high play an important role in the preferential homing of 4 7 memory lymphocytes, which carry memory for intestinal Ags (44) to the gastrointestinal tract and mucosal immune system (28, 42). ␤ Indeed, 7-deficient mice have hypoplastic gut-associated lymphoid tissue (GALT) and a significant reduction in the number of CD4ϩ, CD8ϩ, and plasma cells in the lamina propria (45). There- fore, the ␣ ␤ expression in peripheral memory T cells provides a

4 7 Downloaded from mechanism for the segregation of intestinal from systemic immune responses (42). The chemokine receptor CCR9 has been shown to be expressed ␣ ␤ ϩ on thymocytes and on a subset of memory 4 7 intestinal trafficking CD4 and CD8 PBL. In addition, all small intestinal LPL and intraepithelial lymphocytes (IELs) express CCR9 (9). In this study, we directly compared the expression of CCR9 between http://www.jimmunol.org/ small bowel and colonic LPL and MLN lymphocytes. Although transcript of CCR9 is absent in colon, we found that a small per- FIGURE 6. a, Chemotactic response of freshly isolated small bowel, centage of colonic lymphocytes express CCR9 by flow cytometry. colonic, and PBMCs to various concentrations of TECK. Representative of We show the selective expression of CCR9 on the majority of at least four independent experiments from different donors with similar small bowel lymphocytes as was also shown previously (9) and, in results. In the experiment shown, small bowel and colonic LPMC were addition, CCR9 expression on a much lower percentage of colonic derived from the same donor. b, Chemotaxis of small bowel or colonic ϩ mononuclear cells to optimal concentration of TECK (500 nM). Small lymphocytes. Moreover, we demonstrate that CCR9 lympho- bowel and colonic lymphocytes were incubated with anti-CCR9 or anti- cytes are significantly enriched in MLN draining small bowel vs by guest on September 25, 2021 CCR3 mAb for1hat4°C, or with pertussis toxin (500 ng/ml) for1hat colon. In addition, phenotypic analysis of CCR9-expressing small 37°C before their addition to the chemotaxis chamber. Similar results were bowel LPL reveals that CCR9 is expressed predominantly on ϩ obtained with two different experiments. SB, Small bowel; PTX, pertussis CD3 lymphocytes, although a subset of B and NK cells are also toxin. CCR9ϩ. It was shown previously that CCR9 is expressed on a significant percentage of B cells but not NK cells on PBL (9). Based on three-color flow cytometry, we show in this study that CD3ϩCCR9ϩ and CD3ϩCCR9Ϫ subsets using flow cytometry CCR9 is expressed on CD4 and CD8 small bowel T lymphocytes and analyzed for cytokine production. The percentage of Th1 or and virtually all CD3ϩCCR9ϩ lymphocytes coexpress CD45RO, Th2 cells among CD3ϩCCR9ϩ and CD3ϩCCR9Ϫ small bowel CD69, and CD95. A subset of CD3ϩCCR9ϩ small bowel lym- lymphocytes was similar, although IFN-␥-producing cells were phocytes also coexpress CD25 and HLA-DR. slightly higher in the CD3ϩCCR9ϩ subset and the percentage of The ligand for CCR9, TECK, has been shown to be expressed in cells producing neither IFN-␥ nor IL-4 were higher in the the thymus and small intestine (9, 13, 37). The mTECK was re- CD3ϩCCR9Ϫ subset (Fig. 8). These data show that small bowel ported to be expressed in the thymus by dendritic cells as well as mucosal T cells expressing CCR9 exhibit a diverse cytokine pro- endothelial cells (37). Another report showed mTECK expression

FIGURE 7. TECK is chemotactic for small bowel T lymphocytes. Representation of indicated subsets to TECK-attracted small bowel mononuclear cells. Left, Percentage of each phenotype in the starting population. Migrated cells to TECK (500 nM) were pooled from the lower wells, stained, fixed, and analyzed by flow cytometry and the percentage of each phenotype (right) that migrated to TECK was determined by FACS. A representative experiment of two is shown. The Journal of Immunology 5075 in the thymus and small intestine by epithelial cells (13). In this The precise mechanism by which TECK is involved in the hom- study, we define for the first time the cells expressing TECK in ing of CCR9ϩ lymphocytes to the small bowel mucosa is currently human intestinal tissues. Human TECK is selectively expressed unknown. A likely explanation is that TECK presented on the in the small bowel but not colonic mucosa by a subset of cells in small bowel endothelial cell surface may trigger firm adhesion of ϩ ␣ ␤ intestinal crypts and lamina propria as well as endothelial cells. circulating CCR9 lymphocytes to the vessel wall via 4 7/MAd- In a recent report mTECK mRNA expression was reported to be CAM-1 interactions, and subsequent transmigration into the lam- restricted to villus epithelial cells, the expression beginning at or ina propria as has been shown for other chemokines (20–23). just below the crypt-villus junction, increasing to maximum level TECK expressed by resident small bowel mucosal stromal cells approximately one-third of the way up the villus, and subsequently may help retain these CCR9ϩ T lymphocytes into the small bowel decreasing toward the villus tip (13). Interestingly, mTECK mucosa. It is also tempting to speculate that TECK, which is con- mRNA was detected in the follicle-associated epithelium of the stitutively expressed in the small bowel intestinal crypts, may be murine PP by in situ hybridization. Although we have not directly involved in the homing and retention of small bowel IELs. Indeed, examined hTECK protein expression in human PP, immunohisto- most of small bowel IELs express CCR9 (9), whereas the majority chemistry of small bowel mucosa shows distinct hTECK protein of colonic IELs are CCR9Ϫ (K. A. Papadakis, unpublished data). expression compared with mTECK mRNA expression profile. In Our data show for the first time a phenotypic difference between human tissue, scattered crypt but not villus epithelial cells stain for small bowel and colonic LPMC in humans, based on the selective TECK protein. The reason for the discrepancy in TECK mRNA expression of the chemokine receptor CCR9, which strongly sug- and protein expression in the small bowel mucosa between mouse gests a mechanism for regulated trafficking of intestinal memory T and human is unknown. Importantly, we show that hTECK is ex- cells to the small bowel vs colon. Downloaded from pressed on small bowel but not colonic endothelial cells. In addi- We finally demonstrate that CCR9ϩ small bowel T lymphocytes tion to the expression profile of hTECK protein in intestinal tis- have a diverse cytokine profile, as similar percentages of sorted sues, we demonstrate that TECK chemoattracts small bowel but CD3ϩCCR9ϩ and CD3ϩCCR9Ϫ small bowel lymphocytes pro- not colonic lymphocytes in vitro. Therefore, the selective expres- duce IFN-␥ or IL-4. Distinct patterns of chemokine receptors are sion of TECK in small bowel compared with colon may account acquired by T cells under Th1 (CXCR3, CCR5)- or Th2 (CCR3, ϩ ϩ

␣ ␤ http://www.jimmunol.org/ for the selective recruitment and retention of CCR9 4 7 T cells in CCR8)-polarizing conditions in vitro, which translates into new small bowel lamina propria. The small number of colonic lympho- migratory behavior toward their respective chemokines (24, 32– cytes expressing CCR9, which are predominantly CD3ϩCD4ϩ, may 36). Therefore, the expression of certain chemokines will influence be recruited to the colonic lamina propria in response to other che- the type of T cell immune response in a tissue, for instance, Th1 or mokine(s) and may only coincidentally express CCR9. It has been Th2. However, other chemokines may have a role in tissue-selec- hypothesized that CCR9 expression is induced on naive T lympho- tive recruitment of lymphocytes from the blood (47). Thymus and cytes in the mucosal environment (9). We further propose that CCR9 activation-regulated chemokine and macrophage-derived chemo- induction must be unique to the small bowel mucosal environment. kine, for example, the ligands for CCR4, strongly attract skin- This idea is supported by the finding that draining MLN from small homing but not intestinal memory T cells. The receptor CCR4 is bowel contain a higher percentage of CCR9ϩ lymphocytes than do highly expressed on cutaneous lymphocyte Ag memory CD4ϩ by guest on September 25, 2021 MLN draining colon. However, in naive T lymphocytes CCR9 ex- cells; therefore, CCR4 expression may define a phenotype of pre- pression could not be induced during culture with several cytokines, dominantly skin homing subset of peripheral memory T cells (47). including TGF-␤ or IL-10, which are highly expressed in the mucosal Consistent with these observations, CCR4 expression was virtually environment (46). Therefore, the mechanism by which CCR9 is in- absent in mucosal T cells (K. A. Papadakis, unpublished results). duced or up-regulated by local microenvironmental factors in the Another example, cutaneous T cell-attracting chemokine (CTACK small bowel inductive sites of the mucosal immune system is cur- or CCL27), a recently described chemokine, is expressed in skin rently unknown. Nevertheless, activated CCR9ϩ cells in draining LNs and selectively chemoattracts cutaneous lymphocyte Ag memory may subsequently recirculate and home selectively to intestinal sites T cells (19). that express TECK, such as the small bowel mucosa. Our data suggest that the TECK/CCR9 ligand/receptor pair is important for the selective homing and retention of CCR9ϩ T lymphocytes to the small intestine instead of colon, and provides a mechanism for regional specialization of the mucosal immune system and the segregation of small bowel from colonic immune responses. The CCR9 phenotype is not linked to a specific cytokine profile because both small bowel and colonic lymphocytes as well as sorted CCR9ϩ and CCR9Ϫ small bowel T lymphocytes produce similar Th1 or Th2 cytokines at the single cell level. Consistent with these observations is the finding that CCR9 expression cannot be induced in naive T cells under Th1-, Th2, or T-regulatory 1 polarizing conditions (9). In summary, the preferential expression of the chemokine receptor CCR9 in small bowel compared with colonic lymphocytes,

ϩ the expression of its ligand, TECK, by small bowel but not colonic FIGURE 8. Cytokine profile of CCR9-expressing small bowel CD3 endothelial and stromal cells, and the in vitro migration of small lymphocytes. Freshly isolated small bowel mononuclear cells were stained bowel but not colonic lymphocytes to TECK, suggest that the with 3C3 mAb followed by a cychrome-conjugated goat anti-mouse IgG (HϩL) secondary Ab. Cells were counterstained with CD3-PE and sorted TECK/CCR9 ligand/receptor pair is important for the regional spe- into CD3ϩ CCR9ϩ and CD3ϩ CCR9Ϫ by flow cytometry. Sorted cells cialization of the mucosal immune system. The selective expres- were immediately stimulated with PMA (10Ϫ7 M) and ionomycin (1 ␮g/ sion of a chemokine, such as TECK, in the small intestine could ml)for4hasdescribed in Materials and Methods. Cytokine production at provide the fine-tuning of mucosal T cell trafficking in combina- the single cell level was assessed by intracellular cytokine staining. tion with the expression of the intestinal mucosal homing ligand 5076 CCR9-POSITIVE T CELLS/TECK EXPRESSION IN HUMAN SMALL BOWEL IMMUNITY

␣ ␤ ␣ ␤ 4 7. Therefore, the combination of CCR9/ 4 7 expression may 21. Stein, J. V., A. Rot, Y. Luo, M. Narasimhaswamy, H. Nakano, M. D. Gunn, provide a novel mechanism to segregate small intestinal from co- A. Matsuzawa, E. J. Quackenbush, M. E. Dorf, and U.H. von Andrian. 2000. The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lym- lonic immune responses. The recent identification of the mouse phoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associ- homologs of TECK and CCR9 will permit further examination of ated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J. Exp. Med. 191:61. their role in T cell development and intestinal immunity by selec- 22. Campbell, J. J., J. Hedrick, A. Zlotnik, M. A. Siani, D. A. Thompson, and tively inhibiting their expression. Further understanding of the se- E. C. Butcher. 1998. Chemokines and the arrest of lymphocytes rolling under lective trafficking of T cell to the small bowel based on CCR9 ex- flow conditions. Science 279:381. 23. Peled, A., V. Grabovsky, L. Habler, J. Sandbank, F. Arenzana-Seisdedos, I. Petit, pression will also help us study immune mechanisms such as oral H. Ben-Hur, T. Lapidot, and R. Alon. 1999. The chemokine receptor SDF-1 tolerance and immune-mediated diseases of the small intestine. stimulates integrin-mediated arrest of CD34ϩ cells on vascular endothelium under shear flow. J. Clin. Invest. 104:1199. 24. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997. Selective expression of Acknowledgments the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005. We thank Dr. Phillip Fleshner and Joanne Gainnie for providing speci- 25. Robert, C., and T. S. Kupper. 1999. Inflammatory skin diseases, T cells, and mens, Krystine Nguyen for isolating LPMC, Patricia Lin for flow cytom- immune surveillance. N. Engl. J. Med. 341:1817. etry, Nassim Kassam for help and advice in generating the anti-TECK mAb 26. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector 5A9, and Richard Deem, Carol Landers, and Offer Cohavy for help with functions. Nature 401:708. the figures and statistical analysis. We also thank Loren Karp for critical 27. Randolph, D. A., G. Huang, C. J. L. Carruthers, L. E. Bromley, and reading of the manuscript. D. D. Chaplin. 1999. The role of CCR7 in Th1 and Th2 cell localization and delivery of B cell help in vivo. Science 286:2159. 28. Butcher, E. C., M. Williams, K. Youngman, L. Rott, and M. Briskin. 1999.

References Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209. Downloaded from 1. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565. 29. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matuzawa, L. T. Williams, and 2. Luster, A. D. 1998. Chemokines-chemotactic cytokines that mediate inflamma- H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ che- tion. N. Engl. J. Med. 338:436. mokine have defects in lymphocyte homing and dendritic cell localization. 3. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and J. Exp. Med. 189:451. their role in immunity. Immunity 12:121. 30. Forster, R., E. A. Mattis, E. Kremmer, E. Wolf, G. Brem, and M. Lipp. 1996. A 4. Mantovani, A. 1999. The chemokine system: redundancy for robust outputs. Im- putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid munol. Today 20:254. organs and specific anatomic compartments of the spleen. Cell 87:1037. 5. Liao, F., R. L. Rabin, C. S. Smith, G. Sharma, T. B. Nutman, and J. M. Farber.

31. Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, http://www.jimmunol.org/ 1999. CC-chemokine receptor 6 is expressed on diverse memory subsets of T ␣ M. D. Cooper, J.L Browning, J. D. Sedgwick, and J. G. Cyster. 1999. Lympho- cells and determines responsiveness to macrophage inflammatory protein 3 . toxin ␣/␤ and tumor necrosis factor are required for stromal cell expression of J. Immunol. 162:186. homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 189:403. 6. Carbonnier, A.-S., N. Kohrgruber, E. Kriehuber, G. Stingl, A. Rot, and 32. Sallusto, F., A. Lanzavecchia, and C. R. Mackay. 1998. Chemokines and che- D. Maurer. 1999. Macrophage inflammatory protein 3␣ is involved in the con- mokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. stitutive trafficking of epidermal Langerhans cells. J. Exp. Med. 190:1755. Today 19:568. 7. Ansel, K. M., L. J. McHeyzer-Williams, V. N. Ngo, M.G. McHeyzer-Williams, and J. G. Cyster. 1999. In vivo-activated CD4 T cells upregulate CXC chemokine receptor 5 33. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, and reprogram their response to lymphoid chemokines. J. Exp. Med. 190:1123. S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, and F. Sinigaglia. 1998. 8. Mazzucchelli, L., A. Blaser, A. Kappeler, P. Scha¨rli, J. A. Laissue, M. Baggiolini, Differential expression of chemokine receptors and chemotactic responsiveness and M. Uguccioni. 1999. BCA-1 is highly expressed in Helicobacter pylori- of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129. induced mucosa-associated lymphoid tissue and gastric lymphoma. J. Clin. In- 34. Loetscher, P., M. Uguccioni, L. Bordoli, M. Baggiolini, and B. Moser. 1998. vest. 104:R49. CCR5 is characteristic of Th1 lymphocytes. Nature 391:344. by guest on September 25, 2021 9. Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, 35. Siveke, J. T., and A. Hamann. 1998. Cutting edge: T helper 1 and T helper 2 cells E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al. 1999. Human G protein-coupled respond differentially to chemokines. J. Immunol. 160:550. receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal 36. Sallusto, F., D. Lenig, C. R. Mackay, and A. Lanzavecchia. 1998. Flexible pro- homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required grams of chemokine receptor expression on human polarized T helper 1 and 2 for thymus-expressed chemokine-mediated chemotaxis. J. Exp. Med. 190:1241. lymphocytes. J. Exp. Med. 187:875. 10. Zaballos, A., J. Gutierrez, R. Varona, C. Ardavin, and G. Marquez. 1999. Cutting 37. Vicari, A. P., D. J. Figueroa, J. A. Hedrick, J. S. Foster, K. P. Singh, S. Menon, edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the N. G. Copeland, D. J. Gilbert, N. A. Jenkins, K. B. Bacon, and A. Zlotnik. 1997. receptor for the chemokine TECK. J. Immunol. 162:5671. TECK: a novel CC chemokine specifically expressed by thymic dendritic cells 11. Yu, C. R., K. W. C. Peden, M. B. Zaitseva, H. Golding, and J. M. Farber. 2000. and potentially involved in T cell development. Immunity 7:291. CCR9A and CCR9B: Two receptors for the chemokine CCL25/TECK/Ck␤-15 38. Wilkinson, B., J. J. T. Qwen, and E. J. Jenkinson. 1999. Factors regulating stem that differ in their sensitivities to ligand. J. Immunol. 164:1293. cell recruitment to the fetal thymus. J. Immunol. 162:3873. 12. Campbell, J. J., J. Pan, and E. C. Butcher. 1999. Cutting edge: developmental 39. Targan, S. R., R. L. Deem, M. Liu, S. Wang, and A. Nel. 1995. Definition of a switches in chemokine responses during T cell maturation. J. Immunol. 163:2353. lamina propria T cell responsive state: enhanced cytokine responsiveness of T 13. Wurbel, M. A., J. M. Philippe, C. Nguyen, G. Victorero, T. Freeman, cells stimulated through the CD2 pathway. J. Immunol. 154:664. P. Wooding, A. Miazek, M. G. Mattei, M. Malissen, B. R. Jordan, et al. 2000. The 40. Yang, S. K., L. Eckmann, A. Panja, and M. F. Kagnoff. 1997. Differential and chemokine TECK is expressed by thymic and intestinal epithelial cells and at- regulated expression of C-X-C, C-C, and C-chemokines by human colon epithe- tracts double- and single-positive thymocytes expressing the TECK receptor lial cells. Gastroenterology 113:1214. CCR9. Eur. J. Immunol. 30:262. 41. Reinecker, H. C., E. Y. Loh, D. J. Ringler, A. Mehta, J. L. Rombeau, and 14. Norment, A. M., L. Y. Bogatzki, B. N. Gantner, and M. J. Bevan. 2000. Murine R. P. MacDermott. 1995. Monocyte-chemoattractant protein 1 gene expression in intes- CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regu- tinal epithelial cells and inflammatory bowel disease mucosa. Gastroenterology 108:40. lated following pre-TCR signaling. J. Immunol. 164:639. 42. Butcher, E. C. 1999. Lymphocyte homing and intestinal immunity. In Mucosal 15. Youn, B. S., C. H. Kim, F. O. Smith, and H. E. Broxmeyer. 1999. TECK, an Immunology, P. L. Orga, J. Mestecky, M. E. Lamm, W. Strober, J. Bienestock, efficacious chemoattractant for human thymocytes, uses GPR-9-6/CCR9 as a spe- and J. R. McGhee, eds. Academic Press, San Diego, CA, p. 507. cific receptor. Blood 94:2533. 43. Berlin, C., E. L. Berg, M. J. Briskin, D. P. Andrew, P. J. Kilshaw, B. Holzmann, 16. Fsˇrster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Mu¨ller, E. Wolf, and ␣ ␤ M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing I. L. Weissman, A. Hamann, and E. C. Butcher. 1993. 4 7 integrin mediates functional microenvironments in secondary lymphoid organs. Cell 99:23. lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 74:185. 17. Willimann, K., D. F. Legler, M. Loetscher, R. S. Roos, M. B. Delgado, 44. Rott, L. S., J. R. Rose´, D. Bass, M. B. Williams, H. B. Greenberg, and ␣ ␤ E. C. Butcher. 1997. Expression of mucosal homing receptor 4 7 by circulating I. Clark-Lewis, M. Baggiolini, and B. Moser. 1998. The chemokine SLC is ex- ϩ pressed in T cell areas of lymph nodes and mucosal lymphoid tissues and attracts CD4 cells with memory for intestinal rotavirus. J. Clin. Invest. 100:1204. activated T cells via CCR7. Eur. J. Immunol. 28:2025. 45. Wagner, N., J. Lohler, E. J. Kunkel, K. Ley, E. Leung, G. Krissansen, ␤ 18. Tanaka, Y., T. Imai, M. Baba, I. Ishikawa, M. Uehira, H. Nomiyama, and O. Yoshie. K. Rajewsky, and W. Muller. 1996. Critical role for 7 integrins in formation of 1999. Selective expression of liver and activation-regulated chemokine (LARC) in the gut-associated lymphoid tissue. Nature 382:366. intestinal epithelium in mice and humans. Eur. J. Immunol. 29:633. 46. Kelsall, B., and W. Strober. 1999. Gut-associated lymphoid tissue: antigen han- 19. Morales, J., B. Homey, A. P. Vicari, S. Hudak, E. Oldham, J. Hedrick, R. Orozco, dling and T-lymphocyte responses. In Mucosal Immunology, P. L. Orga, N. G. Copeland, N. A. Jenkins, L.M. McEvoy, and A. Zlotnik. 1999. CTACK, a J. Mestecky, M. E. Lamm, W. Strober, J. Bienestock, and J. R. McGhee, eds. skin-associated chemokine that preferentially attracts skin-homing memory T Academic Press, San Diego, CA, p. 293. cells. Proc. Natl. Acad. Sci. USA 96:14470. 47. Campbell, J. J., G. Haraldsen, J. Pan, J. Rottman, S. Qin, P. Ponath, 20. Warnock, R. A., J. J. Campbell, M. E. Dorf, A. Matsuzawa, L.M. McEvoy, and D. P. Andrew, R. Warnke, N. Ruffing, N. Kassam, et al. 1999. The chemokine E. C. Butcher. 2000. The role of chemokines in the microenvironmental control of T receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T versus B cell arrest in Peyer’s patch high endothelial venules. J. Exp. Med. 191:77. cells. Nature 400:776.