Phenotype and Effector Function of CC Receptor 9-Expressing Lymphocytes in Small Intestinal Crohn's Disease This information is current as of September 29, 2021. Masayuki Saruta, Qi T. Yu, Armine Avanesyan, Phillip R. Fleshner, Stephan R. Targan and Konstantinos A. Papadakis J Immunol 2007; 178:3293-3300; ; doi: 10.4049/jimmunol.178.5.3293 http://www.jimmunol.org/content/178/5/3293 Downloaded from

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Phenotype and Effector Function of CC 9-Expressing Lymphocytes in Small Intestinal Crohn’s Disease1

Masayuki Saruta,2*QiT.Yu,2* Armine Avanesyan,* Phillip R. Fleshner,† Stephan R. Targan,* and Konstantinos A. Papadakis3*

CCL25/CCR9 chemokine ligand/receptor pair has been reported to play an important role in small bowel (SB) immunity and inflammation. We have previously reported an aberrant SB expression of CCL25 in Crohn’s disease (CD) and an increased frequency of CCR9؉ T cells in the peripheral blood of patients with SB inflammatory diseases such as CD and celiac disease. In .this study, we have characterized the phenotype and effector function of CCR9؉ T cells in mucosal lymphoid tissues in CD ؉ We show that CCR9 T cells isolated from mesenteric lymph nodes (MLN) draining CD SB express a more activated Downloaded from phenotype compared with MLN draining normal SB. Stimulation of CCR9؉ T cells isolated from CD SB lamina propria produced more IFN-␥ and IL-17 in response to anti-CD3 or IL-12/IL-18 stimulation compared with those isolated from normal SB. The addition of TL1A to the combination markedly augmented the secretion of IFN-␥, but not IL-17, by CD lamina propria CCR9؉ T cells. CCL25 incubation of CD SB lamina propria lymphocytes and MLN lymphocytes ␤increased their adhesion to VCAM-1/Fc in vitro. Finally, the TCRV␤ analysis of CCR9؉ T cells revealed a diverse TCRV ؉ ؉ repertoire among MLN CCR9 T cells in patients with SB CD. Our data indicate that CCR9 T cells in SB CD are http://www.jimmunol.org/ proinflammatory and support the rationale for the use of CCR9 antagonists for the treatment of human SB CD. The Journal of Immunology, 2007, 178: 3293–3300.

rohn’s disease (CD)4 is a chronic, relapsing and remitting a critical role in immune homeostasis by regulating lymphocyte inflammatory intestinal disorder that can affect any part trafficking to or within lymphoid organs and in peripheral tissues C of the gastrointestinal tract but often involves the small (7–13). They are classified as C, CC, CXC, and CX3C based on bowel (SB), colon, or both (1). Although CARD15/NOD2 gene the positioning of cysteine residues that form two disulfide bonds variants are associated with ileal location of CD (2, 3), the path- (14). mediate their actions through chemokine recep- by guest on September 29, 2021 ways of immune cell trafficking selectively to the SB in CD tors on the surface of target cells. Chemokine receptors are cur- have not been defined adequately. Recruitment of T lympho- rently divided into four families based on the type of chemokine cytes to the intestinal mucosa plays an important role in the that they bind; they are CXCR1 to CXCR6, CCR1 to CCR10, pathogenesis of inflammatory bowel disease (IBD). Chemo- XCR1, or CX3CR1 (15). kines and their receptors as well as adhesion molecules that are CCL25 is selectively expressed in the thymus and small intes- involved in regulating these processes represent novel thera- tine but not colon (9, 10). The only known receptor for CCL25, peutic targets for human IBD (4–6). CCR9, is highly expressed on developing and small ϳ Chemokines constitute a large family of small ( 8–14 kDa), intestinal lamina propria lymphocytes (LPL) and intraepithelial structurally related heparin binding proteins, which are constitu- lymphocytes (9, 10, 16). We and others (9, 10, 12) have proposed tively expressed in lymphoid and extra lymphoid tissues, and play that the CCL25/CCR9 chemokine ligand receptor pair may play an important role in the regional specialization of intestinal immunity and that the coexpression of CCR9 and ␣ ␤ on the cell surface *Inflammatory Bowel Disease Center and Immunobiology Institute, and †Division of 4 7 Colorectal Surgery, Cedars-Sinai Medical Center and the UCLA School of Medicine, may provide a small intestinal “address code” for circulating in- Los Angeles, CA 90048 testinal memory T cells. We previously reported, that in peripheral Received for publication June 27, 2006. Accepted for publication December 27, 2006. blood (PB), CCR9ϩ T lymphocytes were markedly elevated in The costs of publication of this article were defrayed in part by the payment of page patients with SB immune-mediated diseases, including CD, but not charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in patients with purely colonic CD, and that CCL25 expression is altered in inflamed SB but is not expressed in either normal or 1 This work was supported by grants from the Broad Medical Research Program in Inflammatory Bowel Diseases by the Eli and Edythe L. Broad Foundation (to K.A.P.). inflamed colon. These data suggest the potential involvement of ϩ 2 M.S. and Q.T.Y. contributed equally to this work and should be considered as first CCR9 T cells in the pathogenesis of SB CD (11). Furthermore, authors. we have demonstrated that the memory subset of circulating CCR9ϩ ϩ 3 Address correspondence and reprint requests to Dr. Konstantinos A. Papadakis, CD4 T cells in healthy donors has characteristics of mucosal T Cedars-Sinai Medical Center, 8700 Beverly Boulevard, D-4063, Los Angeles, CA 90048. E-mail address: [email protected] lymphocytes since they have an activated phenotype, respond to anti-CD2 stimulation, and exhibit a Th1 or T regulatory 1 cytokine 4 Abbreviations used in this paper: CD, Crohn’s disease; HPF, high power field; IBD, inflammatory bowel disease; LP, lamina propria; LPL, lamina propria lymphocyte; profiles (12). MFI, mean fluorescence intensity; MLN, mesenteric lymph node; PB, peripheral In this study, we have characterized the phenotype and effector blood; SB, small bowel; UC, ulcerative colitis. function of CCR9ϩ T cells in SB mucosal lymphoid tissues in Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 patients with SB CD and compared it with normal controls. We www.jimmunol.org 3294 CCR9ϩ T LYMPHOCYTES IN SMALL INTESTINAL CD Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Phenotypic characteristics of CCR9ϩ T cells from normal and inflamed CD SB. A, LPL lymphocytes were isolated from normal and CD SB and stained for CCR9, CD3, and the indicated surface markers. The ϩ cells were gated on CD3 T cells and analyzed by FACS. The mean per- ϩ FIGURE 2. Phenotype of CCR9 T cells from MLN draining normal centage Ϯ SD of surface markers among CCR9ϩ T cells are shown be- and CD SB. A, MLN lymphocytes draining SB were isolated from five CD tween normal and CD SB (n ϭ 6). A representative FACS analysis of SB and three normal specimens and stained with CCR9, CD3, and the indi- LPL from CD (B) and normal SB (C) is shown. The numbers indicate the ϩ ϩ cated surface markers. The cells were gated on CD3 T cells and analyzed percentage of marker positive cells among CCR9 T cells (right upper ϩ by FACS. The mean percentage Ϯ SD of surface markers among CCR9 panel) and CCR9Ϫ T cells (right lower panel). T cells is shown. A representative FACS analysis of MLN draining CD SB (B) and normal SB (C) is shown. The numbers indicate the percentage of ϩ ϩ marker-positive cells among CCR9 T cells (right upper panel) and show that CCR9 T cells isolated from mesenteric lymph node CCR9Ϫ T cells (right lower panel). (MLN)-draining CD SB have an activated phenotype and those from CD SB lamina propria (LP) exhibit a predominant Th1 and ThIL-17 cytokine profile. CCR9ϩ T cells isolated from MLN- draining SB CD express a polyclonal TCRV␤ repertoire. In addi- Materials and Methods tion, we show that CCL25 triggers the adhesion of CD SB LPL and Patients MLN lymphocytes to VCAM-1/Fc in vitro and therefore could ϩ We enrolled patients with CD, ulcerative colitis (UC), or non-IBD gastro- contribute to the CCR9 trafficking to the SB in inflamma- intestinal disorders who were hospitalized at the Cedars-Sinai Medical tory conditions. Center and underwent intestinal resection. The study was approved by the The Journal of Immunology 3295

(Sigma-Aldrich), 0.1 mg/ml DNase I (Sigma-Aldrich), 50 ␮g/ml gen- tamicin, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 50 ␮g/ml fungizone, in a shaking water bath (100 rpm). The supernatants was collected, filtered through 110-␮m nylon mesh (Spectrum Laboratory Products), and centrifuged at 500 ϫ g for 5 min. The cell pellet was resuspended in 15 ml of solution and was centrifuged at 30 ϫ g for 5 min to remove epithelial and other large cells. The supernatant was removed, and lymphocytes were isolated by separation on Ficoll- Hypaque gradients. The cells were then washed three times with HBSS and resuspended in complete RPMI.

FACS analysis For staining of cell surface Ags, 5 ϫ 105 freshly isolated LPL or MLN lymphocytes were washed twice with PBS supplemented with 0.1% BSA and 0.1% azide and resuspended in 100 ␮l of 10% human Ab serum to block nonspecific Fc binding for 15 min. The cells were incubated with

ϩ the anti-CCR9 mAb 3C3 for 30 min on ice, washed with PBS/BSA/ FIGURE 3. Phenotype of CCR9 T cells in colonic MLN. MLN lym- azide, and incubated with a secondary goat anti-mouse IgG2b-TC for 30 phocytes draining colon were isolated from CD (n ϭ 7), UC (n ϭ 5), and min on ice. The cells were washed again with PBS/BSA/azide and non-IBD (n ϭ 3) surgical specimens stained with CCR9, CD3, and the incubated with mouse IgG for 15 min. FITC- and PE-conjugated mAb indicated surface markers. The cells were gated on CD3ϩ T cells and for surface Ag were used for 30 min. After washing twice, cells were analyzed by FACS. The mean percentage Ϯ SD of surface markers among resuspended in 400 ␮l of 1% paraformaldehyde in PBS and analyzed by Downloaded from ϩ ϫ 4 CCR9 T cells is shown. FACS (BD Biosciences). A total of 3 10 events was routinely col- lected and analyzed using CellQuest software (BD Immunocytometry Systems). Cedars-Sinai Medical Center Institutional Review Board (protocol nos. 3202 and 4254). Analysis of intracellular For cytokine detection at the single-cell level, LPL from inflamed or nor- Abs and reagents mal SB were stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 1 ␮g/ml http://www.jimmunol.org/ ionomycin for 5 h. Brefeldin A (10 ␮g/ml) or monensin (2 ␮M) was added Anti-CD3 and anti-HLA-DR dye-linked mAbs for immunofluorescence to the culture after2hofstimulation to block cytokine secretion. Cells ␤ studies were obtained from Caltag Laboratories. Anti-CD25, -CD69, - 7 were surface stained with CD3-FITC and CCR9-TC, fixed, and permeabil- integrin, -CD40L, -OX40, -CTLA, and -CD45RO dye-linked mAbs ized using Cytofix/Cytoperm solution (Caltag Laboratories). The cells were were obtained from BD Pharmingen. The PE-conjugated anti-cytokine then stained with PE- or AlexaFluor488-conjugated Abs to IFN-␥, IL-10, Abs to IFN-␥, IL-4, IL-5, and IL-10 were from BD Pharmingen. The IL-4, IL-5, or IL-17 or their isotype controls. After an additional wash step, AlexaFluor488 anti-cytokine Ab to IL-17 was from eBioscience. The anti- cells were analyzed by flow cytometry. CCR9 mAb, 3C3, was from Millennium Pharmaceuticals. Recombinant human VCAM-1/Fc chimera (catalog no. 862-VC) was from R&D Sys- Analysis of IFN-␥ and IL-17 production by ELISA tems. IL-12 and IL-18 were purchased from PeproTech. TL1A was from Human Genome Sciences (17). IFN-␥ was quantitated in culture supernatants by amplified sandwich by guest on September 29, 2021 ELISA as reported previously (12). Briefly, 96-well microtiter plates were Isolation of MLN and LP lymphocytes coated overnight with anti-IFN-␥ mAb (BD Pharmingen). After blocking in PBS-BSA, diluted standards (recombinant human IFN-␥; R&D Sys- Lymphocytes were isolated from MLN draining involved SB from CD tems), and samples were added for 24–72 h and detected by a second patients undergoing surgical resection following mechanical disruption and anti-IFN-␥ biotinylated mAb (BD Pharmingen) for 2 h. After washing, release of cells in complete RPMI (RPMI 1640 containing 2 mM L-glu- this biotinylated mAb was detected by streptavidin-alkaline phospha- tamine, 1% nonessential amino acids, 1% sodium pyruvate, 50 ␮g/ml pen- tase (Jackson ImmunoResearch Laboratories) for 30 min, followed by four icillin/streptomycin, and 10% heat-inactivated FCS). MLN-draining nor- phosphate-free washes. Substrate (0.2 mM NADPH; Sigma-Aldrich) was mal SB were isolated from three patients undergoing resection for cancer added for 30 min, then the NADH signal was amplified using 2-propanol (2) or polyposis syndrome (1). We also studied the phenotype of MLN (3%) with iodonitrotetrazoliun violet (1 mM), alcohol dehydrogenase (75 draining isolated from seven CD, five UC, or three non-IBD colonic re- mg/ml), and diaphorase (50 mg/ml, the last three from Sigma-Aldrich), and section specimens. plates were read at 490 nm on an Emax plate reader (Molecular Devices). LPL were isolated from SB CD intestinal specimens or normal SB as Sample concentration was calculated from a standard curve generated by described previously (11). Briefly, the intestinal specimen was washed with our own software (R. Deem). HBSS and the mucosa was dissected away from the underlying layers. The IL-17 was quantitated in culture supernatants by amplified sandwich mucosa layer was incubated in a shaking water bath (100 rpm) in calcium- ELISA. Ninety-six-well microtiter plates were coated overnight with anti- and magnesium-deficient HBSS, containing 1 mM EDTA, 50 ␮g/ml gen- IL-17 mAb (eBioscience). After blocking in assay diluent (eBioscience), tamicin, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 50 ␮g/ml am- diluted standards (recombinant human IL-17; eBioscience) and samples photericin B, with the solution changed every 30 min until the supernatant were added for 24–72 h and detected by a second anti-IL-17 biotinylated was free of epithelial cells. The remaining LP was minced into 1- to 2-mm mAb (eBioscience) for 2 h. After washing, this biotinylated mAb was pieces and was digested for 10 min in RPMI 1640 containing 10% FCS, 0.5 detected by avidin-HRP (eBioscience) for 30 min. Substrate (tetramethyl- mg/ml collagenase B (Boehringer Mannheim), 1 mg/ml hyaluronidase benzidine solution; eBioscience) was added for 15 min followed by 1 M

Table I. IFN-␥ and IL-10 production by CCR9ϩ T cells from normal and CD SBa

% CCR9ϩIFN-␥ϩb MFIb % CCR9ϩIL-10ϩb MFIb

Normal SB (n ϭ 5) 47.8 Ϯ 4 46.5 Ϯ 12 7.2 Ϯ 1.8 40.5 Ϯ 18 CD SB (n ϭ 5) 56 Ϯ 11 57.8 Ϯ 24 8.2 Ϯ 4 40.1 Ϯ 29 p Value 0.1 0.2 0.3 0.49

a LPL were isolated from normal and CD SB as described in Materials and Methods and activated for 5 h with PMA plus ionomycin. After2hofstimulation, brefeldin A (for IFN-␥ staining) or monensin (for IL-10 staining) was added. The cells were stained for CCR9-TC and CD3-FITC and intracellular IFN-␥-PE or IL-10-PE and analyzed by flow cytometry. b The data represent the mean percentage Ϯ SD of cytokine-positive cells and the MFI for IFN-␥ and IL-10 in CCR9ϩ T cells from normal and CD SB. 3296 CCR9ϩ T LYMPHOCYTES IN SMALL INTESTINAL CD

CD3ϩCCR9Ϫ cells, and individual TCR families were evaluated based on T cells showing exclusive staining for either FITC or PE or double staining for both FITC and PE. Thus, 24 individual TCR V␤ families can be analyzed in eight individual tubes. Statistical analysis Differences between the percentage of phenotypic markers or cytokine producing cells between CCR9ϩ and CCR9Ϫ T cells were compared with a t test. Values of p Ͻ0.05 were considered statistically significant. Results CCR9ϩ T cells from MLN draining inflamed SB have an activated phenotype To determine the potential involvement of CCR9ϩ T lymphocytes in SB CD, we analyzed the phenotype of CCR9ϩ T cells isolated from normal and inflamed SB LP. LPL were isolated from normal or CD SB and stained for CD3, CCR9, and for a panel of activation markers and costimulatory molecules and analyzed by FACS. As shown in Fig. 1, we observed no major differences in the pheno- ϩ ϩ FIGURE 4. Expression of IFN-␥ and IL-17 by stimulated CCR9 T type of CCR9 T cells isolated from CD vs normal SB. In both Downloaded from cells from normal and CD SB. SB LPL were isolated as described in Ma- normal and CD SB, most T cells express CCR9 (Fig. 1, B and C), terials and Methods, stimulated with PMA plus ionomycin, and stained for but a small percentage of them coexpress CD25, OX40, surface CCR9, CD3, and intracellular IFN-␥ and IL-17 (A). Representative dot plot CTLA-4, or CD40L. HLA-DR was expressed by a subset of of intracellular IFN-␥ and IL-17 staining from a normal and a CD specimen ϩ ϩ ϩ CCR9 T cells from both normal and CD SB, whereas CD69 was on gated CD3 CCR9 T cells is shown. The mean percentage of IFN-␥ ϩ ϩ highly expressed by most CCR9 T cells from both normal and and IL-17 producing cells among CCR9 T cells in normal and CD SB is ϩ B CD SB (Fig. 1). Most CCR9 T cells in both normal and CD SB http://www.jimmunol.org/ shown in Tables I and II. , Intracellular cytokine staining for IL-10, IL-4, ␤ and IL-5 in normal and SB CD LPL. also expressed CD45RO and 7 integrin (Fig. 1). Therefore, CCR9ϩ T cells from both CD and normal SB show equal levels of activation. ϩ H3PO4 stop solution. Plates were read at 450 nm on an Emax plate reader In contrast, when we analyzed the phenotype of CCR9 T cells (Molecular Devices). from MLN-draining inflamed CD SB, we found a higher percent- ϩ Static adhesion assay age of CCR9 T cells with an activated phenotype compared with MLN-draining normal SB (Fig. 2). The clearest differences among For adhesion assay, 4-mm wells on 18-well glass slides (Cel-Line/Erie ϩ ␮ ␮ CCR9 T cells from inflamed compared with normal SB MLN Scientific) were coated at 4°C with 10 l of VCAM-1/Fc (20 ng in 10 l by guest on September 29, 2021 were seen in the expression of HLA-DR, OX-40, and CD40L. of PBS). After overnight incubation, VCAM-1/Fc solution was aspirated ϩ from the wells, and CCL25 (50 ng in 15 ␮l of PBS) was added to the wells Although the frequency of CD45RO expression by CCR9 T cells for1hat37°C. The wells were blocked with 15 ␮lofFCSfor1hat37°C was lower in MLN compared with SB LP (Figs. 1 and 2), there after extensive washing. LPL or MLN lymphocytes were suspended in were no differences in marker expression in CCR9ϩ T cells be- adhesion buffer (150 mM NaCl, 10 mM HEPES (pH 7.4), 1 mM CaCl2, ϫ 4 tween MLN draining CD and normal SB (Fig. 2). Our data indicate and 1 mM MgCl2) and added to the wells (4.0–4.5 10 cells in 15 ␮l/well). Cells were allowed to adhere for 30 min at 37°C. After washing that T cell activation in MLN-draining SB leads to acquisition ␤ the slides, adherent cells were counted using microscopy. The number of of CCR9 and 7 integrin expression, which would target them adherent cells from five high power fields (HPF) per condition was counted for homing to the SB mucosa. CCR9ϩ T cells isolated from and the mean was calculated. Adhesion of cells stimulated directly with MLN-draining SB appear to have a more activated phenotype in Mn2ϩ was used as positive control. SB CD compared with normal SB. In contrast, we found no ϩ Determination of V␤ families by flow cytometry clear differences in the phenotypic characteristics of CCR9 T cells isolated from MLN draining UC, CD, or normal colon, The IOTest ␤ Mark (TCR V␤ repertoire kit) was obtained from Beckman Coulter. Detection of individual V␤ families was conducted in freshly although there was a trend for increased frequency of OX40 and ϩ harvested MLN lymphocytes from patients with SB CD. A total of 5 ϫ CD40L in UC CCR9 T cells (Fig. 3). 5 10 cells was stained simultaneously with mAbs directed against CD3- ϩ PerCP, CCR9-allophycocyanin, and a set of three Abs directed against CCR9 T cells isolated from inflamed SB have a Th1 and TCRV␤ families. mAbs directed against TCR families were labeled ThIL-17 cytokine profile with either FITC or PE, and the third anti-TCR-directed mAb was la- ϩ beled with PE and FITC. Flow cytometry was performed using the We have previously reported that CCR9 T cells isolated from PB MoFlo (DakoCytomation). Cells were gated on the CD3ϩCCR9ϩ or and normal SB have a prominent Th1 cytokine profile as they

Table II. IL-17, IL-4, and IL-5 production by CCR9ϩ T cells from normal and CD SBa

% CCR9ϩIL-17ϩb % CCR9ϩIL-4ϩb % CCR9ϩIL-5ϩb

Normal SB (n ϭ 4) 10.1 Ϯ 1.7 1.9 Ϯ 0.93* 0.36 Ϯ 0.15* CD SB (n ϭ 9) 10.6 Ϯ 5.0 1.7 Ϯ 1.0† 0.79 Ϯ 0.46† p Value 0.85 0.80 0.17

a LPL were isolated from normal and CD SB as described in Materials and Methods and activated for 5 h with PMA plus ionomycin. After2hofstimulation, brefeldin A and monensin were added. The cells were stained for CCR9-TC and CD3-FITC and intracellular IL-17-AlexaFluor488, or IL-4-PE, or IL-5-PE and analyzed by flow cytometry. b The data represent the mean percentage Ϯ SD of cytokine-positive cells for IL-17, IL-4, and IL-5 among CCR9ϩ T cells .three donors; †, seven donors ,ء .from normal and CD SB The Journal of Immunology 3297

ated with the pathogenesis of several autoimmune diseases, includ- ing experimental colitis. CCR9ϩ T cells from either normal or CD SB LPL contained a higher percentage of IFN-␥-producing cells compared with CCR9Ϫ T cells (data not depicted). The percentage of CCR9ϩ IFN-␥-producing cells were slightly higher in SB CD LPL compared with normal SB LPL (56 Ϯ 11 vs 47.8 Ϯ 4%), but the difference was not statistically significant (Table I and Fig. 4A). The percentage of CCR9ϩIL-10ϩ, CCR9ϩIL-4ϩ, and CCR9ϩ IL-5ϩ cells among these two groups was also similar and signif- icantly lower compared with the percentage of IFN-␥-producing cells (Tables I and II and Fig. 4B). The percentage of CCR9ϩIL-17-producing cells were similar in SB CD LPL compared with normal SB LPL (10.6 Ϯ 5.0 vs 10.1 Ϯ 1.7, p ϭ NS) (Table II and Fig. 4A). The CCR9ϩIL-17ϩ T cells were largely distinct from the IFN-␥-producing cells, whereas a small percentage of CCR9ϩ T cells were IFN-␥ϩIL-17ϩ double- positive cells (2.6 Ϯ 1.4% in CD LPL vs 3.5 Ϯ 1.8% in normal SB LPL; p ϭ 0.37) (Fig. 4A). Our data indicate that CCR9ϩ T cells Downloaded from isolated from normal and CD SB have a dominant Th1 cytokine profile, but there were no overall differences in the percentage of IFN-producing cells or the mean fluorescence intensity (MFI) of intracellular IFN-␥ staining between CD and normal SB CCR9ϩ T cells when they are maximally activated with PMA plus iono- mycin. In addition, ϳ10% of CCR9ϩ T cells produce IL-17 and most are distinct from IFN-␥-producing cells in both normal http://www.jimmunol.org/ and CD SB. ϩ ␥ FIGURE 5. A, IFN-␥ production by CCR9 T cells from normal and We next analyzed the accumulation of IFN- protein in culture ϩ CD SB lymphocytes. SB LPL were isolated, and CD3ϩCCR9ϩ T cells supernatants from sorted CCR9 T cells isolated from CD vs nor- ϩ were sorted by FACS and stimulated with plate-bound anti-CD3 and sol- mal SB LPL. As shown in Fig. 5A, CCR9 T cells isolated from uble anti-CD28, soluble anti-CD2 and anti-CD28, or IL-12 (1 ng/ml) and CD LPL secrete significantly more IFN-␥ compared with CCR9ϩ IL-18 (50 ng/ml) with or without TL1A (100 ng/ml) or left unstimulated. T cells isolated from normal LPL when activated with anti-CD3 ␥ Forty-eight hours later, supernatants were collected and analyzed for IFN- plus anti-CD28 Abs or with the cytokines IL-12 and IL-18. Inter- content by ELISA. The mean Ϯ SD of IFN-␥ concentration from four estingly, the addition of TL1A, which was recently shown to syn- by guest on September 29, 2021 donors from inflamed and normal SB is shown. B, IL-17 production by ϩ ergize with IL-12 and IL-18 to enhance IFN-␥ production by CCR9 T cells from normal and CD SB lymphocytes. Supernatants col- ϩ lected from the experiments described in A were also analyzed for IL-17 CCR9 PB and LP T cells (13), significantly enhanced the secre- ϩ protein content by ELISA. tion of IFN-␥ by CCR9 T cells isolated from inflamed CD SB compared with CCR9ϩ T cells isolated from normal SB (Fig. 5A). The maximum amount of IFN-␥ released from CCR9ϩ T cells (SB secrete large amounts of IFN-␥, whereas a small subset of CD, 141 ng/ml vs normal SB, 29 ng/ml, n ϭ 4, p ϭ 0.01) was ϩ CCR9ϩIFN-␥Ϫ PB T cells also produces IL-10 (10, 12). There- observed when CCR9 T cells were stimulated with IL-12 plus fore, we examined whether CCR9ϩ T cells isolated from CD SB IL-18 plus TL1A (Fig. 5A). LP have a predominant Th1 cytokine profile and whether they also Furthermore, we analyzed the accumulation of IL-17 protein in produce IL-17 because the IL-23/IL-17 pathway has been associ- culture supernatants from sorted CCR9ϩ T cells isolated from CD

FIGURE 6. CCL25 triggers the adhesion of SB CD LPL and MLN lymphocytes to immobilized VCAM-1/Fc in vitro. Isolated SB CD LPL and MLN lymphocytes were added to 4-mm VCAM-1/Fc- coated well glass slides as described in Materials and Methods. After washing, the adherent cells were counted in five randomly selected HPF with micros- copy. The data shown represent the mean number of adherent cell per HPF Ϯ SD. One representative ex- periment from three different donors is shown. 3298 CCR9ϩ T LYMPHOCYTES IN SMALL INTESTINAL CD

FIGURE 7. CCR9ϩ T cells from MLN- draining CD SB express a diverse TCRV␤ repertoire. MLN lymphocytes were isolated from patients with active SB CD (n ϭ 5), stained with CCR9-allophycocyanin, CD3- PerCP, and a combination of PE, FITC, or PE plus FITC-conjugated Ab against 24 TCRV␤ families, and analyzed by flow cy- indicates significant differences ء .tometry (p Ͻ 0.05) between CCR9ϩ and CCR9Ϫ T cells. Downloaded from

vs normal SB LPL. As shown in Fig. 5B, CCR9ϩ T cells isolated CCR9Ϫ T cells (Fig. 7). These changes in the frequency of V␤1, from CD LPL secrete significantly more IL-17 compared with V␤11, and V␤13.6 were significant at the p Ͻ 0.05 level. CCR9ϩ T cells isolated from normal LPL, especially when stim- ulated with anti-CD2 plus anti-CD28 Abs or anti-CD3 plus anti- http://www.jimmunol.org/ CD28 Abs. Unlike with IFN-␥ production, the addition of TL1A Discussion had no effect on IL-17 production by CCR9ϩ T cells isolated from In this study, we have examined the phenotype and cytokine pro- file of mucosal CCR9ϩ T cells in patients with SB CD. We found inflamed CD SB or normal SB (Fig. 5B). The average amount of ϩ IL-17 released from CCR9ϩ T cells in SB CD was 15 Ϯ 7.6 vs that a higher percentage of CCR9 T cells isolated from MLN 1.8 Ϯ 0.8 ng/ml in normal SB (n ϭ 4, p ϭ 0.01) when CCR9ϩ T draining CD SB, but not the LP, express the activation markers cells were stimulated with anti-CD2 plus anti-CD28 Abs (Fig. 5B). OX40, CD40L, and HLA-DR compared with MLN-draining nor- mal SB. Although the percentage of CCR9ϩ T cells was lower in MLN-draining CD SB than normal SB in our previous study (11),

TECK/CCL25 triggers the adhesion of SB CD MLN lymphocytes by guest on September 29, 2021 there appears to be a variability in the detected frequency of CCR9 and LPL to VCAM-1 expression among SB MLN T cells in CD possibly related to the We have previously reported altered CCL25 expression in in- stage of the inflammatory process or the concomitant use of med- flamed CD SB compared with normal SB by immunohistochem- ications. Nevertheless, CCR9ϩ T cells isolated from MLN drain- ϩ istry and also an increased frequency of CCR9 T cells in the ing inflamed CD SB have a more activated phenotype than circulation of patients with SB CD. Since chemokines play a crit- those isolated from normal SB MLN, whereas CCR9ϩ T cells ical role not only in lymphocyte migration but also in integrin from both CD and normal SB show equal levels of activation. activation (18–20), we hypothesized that the aberrant CCL25 ex- The fact that we found no clear differences in the phenotypic ϩ pression may contribute to SB homing of effector CCR9 T cells characteristics of CCR9ϩ T cells isolated from MLN-draining to the SB through integrin activation, endothelial cell adhesion, UC, CD, or normal colon indicates that these differences are and subsequent transmigration into the LP. Therefore, we exam- specific to SB CD. ined the ability of CCL25 to trigger CD SB LPL or MLN lym- We have previously shown that CCR9ϩ T cells isolated from phocytes to adhere to VCAM-1. As shown in Fig. 6, CCL25 trig- normal SB exhibit a prominent Th1 cytokine profile since they gered adhesion of both SB CD LPL and MLN lymphocytes to mainly produce IFN-␥ (10). In this study, we demonstrate that LP VCAM-1/Fc-coated slides in a static adhesion assay. CCL25-in- CCR9ϩ T cells isolated from CD as well as normal SB produced ϩ duced adhesion was as robust as that observed with Mn2 , which mainly IFN-␥ but little IL-10 as assessed by intracellular cytokine was used as positive control. staining following in vitro stimulation with PMA plus ionomycin. ϩ ϩ ϩ However, when LP CCR9 CD3 T cells isolated from inflamed CCR9 T cells from the MLN draining CD SB display a diverse CD SB were stimulated with anti-CD3 Abs or IL-12 and IL-18, ␤ TCRV repertoire they accumulated significantly more IFN-␥ in culture supernatants We next hypothesized that CCR9ϩ T cells may expand secondary compared with normal SB. Similar findings we observed in regards to specific antigenic stimulation in draining MLN and therefore to IL-17 production by CCR9ϩ T cells. Approximately 10% of LP may be associated with a restricted expression of TCRV␤ families. CCR9ϩ T cells in both CD and normal SB produced IL-17, and We used the IOTest ␤ Mark kit (Beckman Coulter), a flow cy- most were distinct from IFN-␥-producing cells. However, when tometry-based assay for quantitative analysis of the TCR V␤ rep- CCR9ϩ T cells isolated from inflamed CD SB were stimulated in ertoire of human T lymphocytes, to determine the frequency of 24 vitro with anti-CD3 or anti-CD2 Abs, they accumulated signifi- V␤ families covering ϳ70% of the TCR repertoire of human T cantly more IL-17 in culture supernatants compared with normal lymphocytes. We found that SB CD MLN lymphocytes express a SB (Fig. 5B). diverse set of TCRV␤ among both CCR9ϩ and CCR9Ϫ T cells We reported previously that TL1A, a newly discovered cyto- (Fig. 7). Interestingly, subsets of TCRV␤, such as V␤1, V␤11, and kine, synergizes with IL-12 and IL-18 to enhance IFN-␥ in human V␤13.6, were overrepresented among CCR9ϩ compared with T cells, and particularly in PB and SB CCR9ϩ T cells (13, 17). In The Journal of Immunology 3299 this study, we also demonstrate that addition of TL1A to IL-12/ small molecule CCR9 antagonist CCX282 (Trafficet-EN; IL-18 cytokine combination markedly enhanced secretion of Chemocentryx) in the treatment of experimental ileitis (26). IFN-␥ by CCR9ϩ T cells from inflamed CD SB, indicating an The presence of activated CCR9ϩ T cells with enhanced Th1- important role of TL1A/DR3 interactions in enhancing Th1 im- and ThIL-17-producing capacity in human SB CD supports the mune responses in CD from cytokine stimulated CCR9ϩ T cells. rationale for the use of selective CCR9 antagonists for the treat- Recently, a similar role of TL1A has been proposed in murine ment of SB CD. models of SB CD (21). In marked contrast to its effect on IFN-␥ production, TL1A had no effect on IL-17 production by cytokine- stimulated CCR9ϩ LP T cells. Acknowledgment Lymphocyte homing to peripheral tissues is controlled by the We thank Martin R. Hodge (Millennium Pharmaceuticals) for providing us combinatorial interactions between cell integrins and their re- with the 3C3 anti-CCR9 mAb. spective ligands expressed on endothelial cells (7). Selective homing to mucosal lymphoid tissues is mediated through inter- ␣ ␤ actions between 4 7 and MAdCAM-1 (22). The combined ex- Disclosures ␣ ␤ pression of 4 7/CCR9 by intestinal homing T lymphocytes The authors have no financial conflict of interest. may further define SB vs colonic mucosal T cell trafficking through CCL25 (selectively expressed in the SB) triggering of integrin activation from CCR9 engagement (9, 10). Therefore, References expression of CCR9 by mucosal homing T cells may be used to 1. Podolsky, D. K. 2002. Inflammatory bowel disease. N. Engl. J. Med. 347: Downloaded from distinguish SB from colonic immune responses (12). CCL25 is 417–429. selectively expressed in the thymus and SB endothelial cells, 2. Abreu, M. T., K. D. Taylor, Y. C. Lin, T. Hang, J. Gaiennie, C. J. Landers, E. A. Vasiliauskas, L. Y. Kam, M. Rojany, K. A. Papadakis, et al. 2002. Muta- intestinal crypts, and a subset of LP cells (9, 10). In contrast, tions in NOD2 are associated with fibrostenosing disease in patients with Crohn’s there is no expression of CCL25 in normal or inflamed colon disease. Gastroenterology 123: 679–688. (11). The expression of CCL25 by SB endothelial cells could 3. Ahmad, T., A. Armuzzi, M. Bunce, K. Mulcahy-Hawes, S. E. Marshall, T. R. Orchard, J. Crawshaw, O. Large, A. de Silva, J. T. Cook, et al. 2002. The mo- provide the chemokine signal for the firm arrest of CCR9-ex- lecular classification of the clinical manifestations of Crohn’s disease. Gastro- http://www.jimmunol.org/ pressing T cells rolling on SB vascular beds and subsequent enterology 122: 854–866. ␣ extravasation into the LP explaining the preferential recruit- 4. Lobb, R. R., and M. E. Hemler. 1994. The pathophysiologic role of 4 integrins ϩ in vivo. J. Clin. Invest. 94: 1722–1728. ment of CCR9 T cells to the SB. A previous study has also 5. Ghosh, S., E. Goldin, F. H. Gordon, H. A. Malchow, J. Rask-Madsen, reported the aberrant expression of CCL25 in the liver in pa- P. Rutgeerts, P. Vyhnalek, Z. Zadorova, T. Palmer, and S. Donoghue. 2003. tients with primary sclerosing cholangitis. CCL25 could trigger Natalizumab for active Crohn’s disease. N. Engl. J. Med. 348: 24–32. adhesion of liver-infiltrating lymphocytes that expressed CCR9 6. Sandborn, W. J., J. F. Colombel, R. Enns, B. G. Feagan, S. B. Hanauer, I. C. Lawrance, R. Panaccione, M. Sanders, S. Schreiber, S. Targan, et al. 2005. to immobilized MAdCAM-1 in vitro, establishing a link be- Natalizumab induction and maintenance therapy for Crohn’s disease. N. Engl. tween IBD and primary sclerosing cholangitis through CCR9ϩ J. Med. 353: 1912–1925. T cell-mediated inflammation (23). In this study, we demon- 7. Butcher, E. C., M. Williams, K. Youngman, L. Rott, and M. 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