And Th1 Or T-Regulatory 1 Cytokine Profile Lymphocytes with Mucosal T

CC Chemokine Receptor 9 Expression Defines a Subset of Peripheral Blood Lymphocytes with Mucosal T Cell Phenotype and Th1 or T-Regulatory 1 Cytokine Profile This information is current as of September 28, 2021. Konstantinos A. Papadakis, Carol Landers, John Prehn, Elias A. Kouroumalis, Sofia T. Moreno, Jose-Carlos Gutierrez-Ramos, Martin R. Hodge and Stephan R. Targan J Immunol 2003; 171:159-165; ; doi: 10.4049/jimmunol.171.1.159 Downloaded from http://www.jimmunol.org/content/171/1/159

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CC Chemokine Receptor 9 Expression Deﬁnes a Subset of Peripheral Blood Lymphocytes with Mucosal T Cell Phenotype and Th1 or T-Regulatory 1 Cytokine Proﬁle1

Konstantinos A. Papadakis,2* Carol Landers,* John Prehn,* Elias A. Kouroumalis,† Soﬁa T. Moreno,* Jose-Carlos Gutierrez-Ramos,‡ Martin R. Hodge,‡ and Stephan R. Targan2*

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9؉ lymphocytes in blood from normal donors. A subset of CCR9؉ T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and ؊ ؉ ؉ OX-40. In contrast to CCR9 , CCR9 CD4 peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce Downloaded from high levels of IFN-␥ and IL-10. IL-10-producing cells were exclusively detected within the CCR9؉ subset of CD4؉ T cells by intracellular staining and were distinct from IL-2- and IFN-␥-producing cells. Moreover, memory CCR9؉CD4؉ lymphocytes -respond to CD2 stimulation with proliferation and IFN-␥/IL-10 production, whereas memory CCR9؊CD4؉ cells were unrespon sive. In addition, memory CCR9؉CD4؉ T cells support Ig production by cocultured CD19؉ B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9؉CD4؉ T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the http://www.jimmunol.org/ cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans. The Journal of Immunology, 2003, 171: 159Ð165.

hemokines and their receptors play a critical role in the potential to home to lymphoid organs and CCR7-effector memory migration of different types of leukocytes, including na- T cells with the potential to migrate to inflamed tissue (8). CXCR5 C ive and memory/effector T lymphocytes, into microana- expression on memory T cells defines a subset that localizes to B tomic compartments of lymphoid organs and peripheral tissues cell follicles and germinal centers and supports Ig production by (1–5). T cells in peripheral blood (PB)3 express a diverse set of cocultured B cells (9, 10), whereas CCR4 and CCR8 are highly by guest on September 28, 2021 chemokine receptors, and studies in the last few years have sup- expressed by human CD4ϩCD25ϩ-regulatory T cells (11). Other ported the notion that the patterns of chemokine receptors charac- chemokine receptors are preferentially expressed by different Th terize functional T cell subpopulations. Naive T cells, which pref- subsets; CCR3 is expressed predominantly by Th2 cells, and erentially migrate to lymph nodes and Peyer’s patches, express CCR5 or CXCR3 are expressed by Th1 cells (12–15). Expression CXCR4, the receptor for stromal cell-derived factor1/CXC ligand of other chemokine receptors by peripheral blood (PB) T cells 12, and CCR7, the receptor for Epstein-Barr virus-induced mole- may define a tissue-specific homing potential, such as CCR4 or cule1 ligand chemokine/C-C chemokine ligand (CCL) 19, and sec- CCR10, which are preferentially expressed by cutaneous lym- ondary lymphoid organ chemokine/CCL21. Mice deficient in phocyte Agϩ-skin homing T cells (16, 17), and CCR9, which is CCR7 or secondary lymphoid organ chemokine/CCL21 have de- ␣ ␤ ϩ expressed by 4 7 -intestine-homing lymphocytes (18–21). fective homing of naive T cells to secondary lymphoid organs (6, These data suggest an important role of certain chemokines and 7). Based on the expression of CCR7, memory T cells can be their receptors in regulating the homing of specific T cell sub- further distinguished into CCR7ϩ central memory T cells with the sets into microanatomic compartments of lymphoid organs and peripheral tissues (4). *Burns and Allen Research Institute and Inflammatory Bowel Disease Center, Ce- We and others have proposed that the chemokine thymus-ex- dars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Los Angeles, pressed chemokine (TECK)/CCL25 and its receptor CCR9 may CA 90048; †Univerity Hospital of Heraklion, University of Crete, Heraklion, Crete, Greece; and ‡Millennium Pharmaceuticals, Cambridge, MA 02139 play an important role in the regional specialization of intestinal immunity and that the combined expression of CCR9 and ␣ ␤ on Received for publication December 20, 2002. Accepted for publication April 4 7 18, 2003. the cell surface may provide a small intestinal address code for The costs of publication of this article were defrayed in part by the payment of page circulating intestinal memory T cells (20, 21). CCR9 is expressed charges. This article must therefore be hereby marked advertisement in accordance on a small subset of PB CD4ϩ (2–4%) and CD8ϩ T cells, most of with 18 U.S.C. Section 1734 solely to indicate this fact. ␣ ␤ which coexpress the mucosal homing ligand 4 7 (18). Here, we 1 This work is supported by Grants DK-46763 and DK-56328 from the National ϩ ϩ Institutes of Health (to S.R.T.) and by a Career Development Award from the Crohn’s show that circulating CCR9 CD4 T cells from normal donors and Colitis Foundation of America (to K.A.P.). have characteristics of mucosal T cells in terms of an activated 2 Address correspondence and reprint requests to Dr. Konstantinos A. Papadakis, phenotype, proliferative response to anti-CD2 stimulation, a Th1 Cedars-Sinai Medical Center, 8700 Beverly Boulevard, D-4063, Los Angeles, CA or T-regulatory 1 (Tr1) cytokine profile, and support for Ig pro- 90048. E-mail address: [email protected] or Dr. Stephan R. Targan, 8700 Beverly Boulevard, D-4063, Los Angeles, CA 90048. E-mail address: [email protected] duction by cocultured B cells. This T cell subset may provide a 3 Abbreviations used in this paper: PB, peripheral blood; CCL, C-C chemokine li- peripheral window to small intestinal immunity in humans and a gand; TECK, thymus-expressed chemokine; Tr1, T-regulatory 1; TC, tricolor. tool to study Ag specificity and cytokine profile of effector and

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 160 CIRCULATING CCR9ϩ LYMPHOCYTES HAVE MUCOSAL T CELL CHARACTERISTICS regulatory cells in small bowel inflammatory diseases and in oral Statistical analysis tolerance. Differences between the percentage of phenotypic markers expressed between CCR9ϩ and CCR9Ϫ T cells were compared with a paired t test. p Ͻ Materials and Methods 0.05 was considered statistically significant. Abs and reagents Results Anti-CD3, -CD4, -CD8, -HLA-DR dye-linked mAbs for immunofluores- ϩ cence studies were obtained from Caltag (Burlingame, CA). Anti-CD25, CCR9 PB T cells contain a higher fraction expressing surface ␤ Ϫ -CD69, - 7 integrin, -CD40L, -OX-40, -CTLA, -CD27, -CD71, -CD62L, activation and costimulatory molecules than does the CCR9 and -CD45RO dye-linked mAbs were obtained from BD PharMingen (San subset Diego, CA). The PE- or FITC-conjugated anti-cytokine Abs to IFN-␥, IL-4, and IL-10 were from BD PharMingen. The FITC-conjugated anti- We have recently shown that patients with small intestinal inflam- IL-2 Ab was from Caltag. PWM was from Sigma-Aldrich (St. Louis, MO). matory diseases, such as Crohn’s and celiac disease, have a higher The anti-CCR9 mAb, 3C3, was from Millennium Pharmaceuticals percentage of circulating CCR9ϩCD4ϩ T cells than do patients (Cambridge, MA). with colonic Crohn’s or normal healthy donors; therefore, this cell Cell isolation, sorting, and FACS analysis subset is potentially involved in these small intestinal inflammatory processes. To gain insight into the functional aspects of the PBLs were isolated from normal healthy volunteers by separation on Fi- ϩ coll-Hypaque gradients. The cells were subsequently washed three times CCR9 PB T cells from normal donors, we first examined the with HBSS and resuspended in RPMI 1640 containing 2 mM L-glutamine, expression of several activation markers and costimulatory mole- 1% nonessential amino acids, 1% sodium pyruvate, 50 ␮g/ml penicillin- cules on CCR9ϩ PB T cells. As shown in Fig. 1 and Table I, more streptomycin, and 10% heat-inactivated FCS. The cells were stained with CCR9ϩ T cells express CD4 and CD8 and the activation markers Downloaded from anti-CCR9 mAb (3C3, IgG2b) followed by a secondary PE- or tricolor Ϫ (TC)-conjugated goat anti-mouse IgG2b mAb (Caltag). The cells were CD25, CD69, CD71, and HLA-DR than do CCR9 T cells. Sim- washed and incubated with mouse IgG for 15 min and subsequently stained ilarly, the fraction of cells expressing OX-40 and CD40L was with FITC-conjugated anti-CD4 mAb. After staining, the cells were sorted higher in CCR9ϩ than in CCR9Ϫ T cells. The CD27ϩ fraction was using FACSVantage (BD Biosciences, San Jose, CA) to isolate similar in the two cell subsets. Most CCR9ϩ and CCR9Ϫ T cells CD4ϩCCR9ϩ and CD4ϩCCR9Ϫ cells (purity, Ͼ99%). In some experiments, cells were stained with anti-CCR9/TC-conjugated goat anti-mouse coexpress CD62L, but the positive fraction was lower for circu-

ϩ Ϫ http://www.jimmunol.org/ IgG2b mAb, PE-conjugated anti-CD45RO, and FITC-conjugated anti-CD4 lating CCR9 compared with CCR9 T cells. Additionally, more mAb and sorted into CD4ϩCD45ROϩCCR9ϩ, CD4ϩCD45ROϪCCR9ϩ, and CD4ϩCD45ROϩCCR9Ϫ cells (purity, Ͼ98%). For staining of cell surface Ags, 5 ϫ 10 5 freshly isolated PBL 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 nonspeciﬁcFc binding for 15 min. The cells were incubated with the anti-CCR9 mAb 3C3 for 30 min on ice, washed with PBS-BSA-azide, and incubated with a secondary goat anti-mouse IgG2b-TC for 30 min on ice. The cells were washed again with PBS-BSA-azide and incubated with mouse IgG for 15

min. FITC- and PE-conjugated mAb for surface Ag were used for 30 min. by guest on September 28, 2021 After two washings, cells were resuspended in 400 ␮l of 1% paraformal- dehyde in PBS and analyzed by FACS (BD Biosciences, San Jose, CA); 3 ϫ 104 events were routinely collected and analyzed using CellQuest software (BD Biosciences). Cytokine detection Sorted CD4ϩCCR9ϩ and CD4ϩCCR9Ϫ T cells or CD4ϩCD45ROϩCCR9ϩ, CD4ϩCD45ROϪCCR9ϩ, and CD4ϩCD45ROϩCCR9Ϫ cells were stimulated with plate-bound anti-CD3 (OKT3, 1 ␮g/ml; American Type Culture Collec- tion, Manassas, VA) with or without soluble anti-CD28 (clone 9.3, 2 ␮g/ml) or with soluble anti-CD2 Abs (clones CB6 and GD10 at 1 ␮g/ml, a gift from C. Benjamin, Biogen, Cambridge, MA) with or without anti-CD28, and cytokine production was measured in the 24- and 72-h culture supernatants by ELISA using matched pairs of Abs specific for IL-10 and IFN-␥. For cytokine detection at the single-cell level, sorted CD4ϩ T cell subsets were stimulated with 50 ng/ml PMA (Sigma) and 1 ␮g/ml ionomycin for 5 h. Brefeldin A (10 ␮g/ml) was added to the culture after2hofstimulation to block cytokine secretion. Cells were fixed and permeabilized using Cytofix-Cytoperm solu- tion (Caltag) and stained with PE- and FITC-conjugated control Abs or mAbs to IL-2, IFN-␥, IL-4, and IL-10. Proliferation assays Sorted CD4ϩCCR9ϩ and CD4ϩCCR9Ϫ T cells or CD4ϩCD45ROϩCCR9ϩ and CD4ϩCD45ROϩCCR9Ϫ cells were stimulated with plate-bound anti-CD3 (1 ␮g/ml) with or without soluble anti-CD28 (2 ␮g/ml) or with soluble anti- CD2 (1 ␮g/ml) with or without anti-CD28 and pulsed with 1 ␮Ci/well [3H]thymidine for the last 16 h of a 96-h culture. Radioactivity was analyzed FIGURE 1. Expression of activation and costimulatory molecules in on a scintillation counter. freshly isolated PB CCR9ϩ T lymphocytes. Freshly isolated PBL from Ab production normal donors were stained with 3C3/IgG2b-TC secondary Ab, anti-CD3, ϩ ϩ ϩ ϩ ϩ Ϫ and Ab against several activation and costimulatory molecules and ana- PB T cells (CD4 CD45RO CCR9 or CD4 CD45RO CCR9 ) and B ϩ ϩ lyzed by FACS. The cells were gated on CD3 lymphocytes. The percent- cells (CD19 ) were sorted by FACS and cocultured in round-bottom 96- ϩ 5 ages in the quadrants represent the percent of CCR9 (upper right)or well plates at 10 cells/well each of T and B cells in the absence of T cell Ϫ activation stimuli or cytokines for 11 days. B cells stimulated with PWM CCR9 (lower right) T lymphocytes that express each cell surface marker (5 ␮g/ml) were used as positive control. IgM, IgG, and IgA contents in the shown in each dot plot. Representative data of three to five different donors culture supernatants were determined by ELISA. examined. The Journal of Immunology 161

Table I. Phenotypic markers on CCR9ϩ and CCR9Ϫ T cells in peripheral blooda

CCR9ϩ T Cells CCR9Ϫ T Cells Marker (% Ϯ SD) (% Ϯ SD)

ءCD4 76 Ϯ 965Ϯ 10 ءءCD8 49 Ϯ 11 29 Ϯ 9 ءءCD25 26 Ϯ 79Ϯ 2 ءءCD69 17 Ϯ 6 1.5 Ϯ 1 ءءCD71 25 Ϯ 93Ϯ 3 ءءHLA-DR 48 Ϯ 16 18 Ϯ 6 ءءOX-40 (CD134) 28 Ϯ 11 6 Ϯ 5 ءءCD40L (CD154) 15 Ϯ 71Ϯ 1.8 CTLA-4 (CD152) 16 Ϯ 11 2 Ϯ 1.5 CD27 92 Ϯ 591Ϯ 6 ءCD62L 78 Ϯ 488Ϯ 4 ءءءCD45RO 68 Ϯ 11 45 Ϯ 8 ءء Ϯ Ϯ ␤ 7 integrin 73 11 50 6 a Freshly isolated PBMC were stained with 3C3/IgG2b-TC secondary Ab, CD3- FITC, or CD3-PE and indicated cell surface markers and analyzed by FACS. The ϩ Ϯ

cells were gated on CD3 lymphocytes. Data represent the mean percent SD of Downloaded from CCR9ϩ or CCR9Ϫ T cells expressing the indicated cell surface markers from three to ﬁve different donors examined. p Ͻ 0.05 between the mean percentage of each ,ءءء ,p Ͻ 0.01 ,ءء ,p Ͻ 0.001 ,ء phenotypic marker expressed in CCR9ϩ vs CCR9Ϫ T cells.

ϩ ϩ ␤ ϩ CCR9 T cells were CD45RO and 7 integrin as compared http://www.jimmunol.org/ with the CCR9Ϫ T cells, as previously described (see also Fig. 1 FIGURE 2. Proliferative responses of sorted CCR9ϩand CCR9Ϫ CD4ϩ and Table I). Collectively, our data show that a subset of circulat- T cells to IL-2, anti-CD3, or anti-CD2 stimulation. A, PBL were stained ing CCR9ϩ T cells have a phenotype associated with activation. ϩ ϩ ϩ with 3C3/IgG2b-PE secondary Ab and CD4 Abs and sorted into two cell Addition of CD4 and CD8 percentages of CCR9 T cells gives subsets, CD4ϩCCR9ϩ and CD4ϩCCR9Ϫ. Sorted cells (105/wells) were ϩ Ͼ100% (see Fig. 1 and Table I), indicating that a subset of CCR9 plated in flat-bottom 96-well plates coated with anti-CD3 (OKT3, 1 ␮g/ T cells must be double-positive T cells, which is consistent with ml). Under other conditions, cells were incubated with IL-2 (200 U/ml), their activation phenotype (22). anti-CD2 (1 ␮g/ml), and anti-CD28 (2 ␮g/ml). Proliferation was determined at day 4, with [3H]thymidine added for the last 16 h of culture. B, ϩ Ϫ ϩ ϩ Ϫ ϩ Proliferative responses of CCR9 and CCR9 CD4 T cells Proliferative responses of memory (m) CCR9 and CCR9 CD4 T cells by guest on September 28, 2021 To determine the activation requirement of PB CCR9ϩCD4ϩ T to anti-CD3 or anti-CD2 stimulation. Cells were incubated with 3C3/ ϩ ϩ ϩ Ϫ IgG2b-TC secondary Ab, -CD4-FITC, and -CD45RO-PE; sorted into cells, sorted CD4 CCR9 and CD4 CCR9 T cells were stim- ϩ ϩ ϩ ϩ ϩ Ϫ Ϯ Ϯ CD4 CD45RO CCR9 , CD4 CD45RO CCR9 ; and plated at a con- ulated with IL-2, or anti-CD3 CD28, or anti-CD2 CD28, and centration of 5 ϫ 104 cells/well. Proliferation was determined at day 4, proliferation of each cell subset was analyzed. As shown in Fig. with [3H]thymidine added for the last 16 h of culture. 2A, CCR9ϩCD4ϩT cells were more responsive to IL-2 or anti- CD3 stimulation alone than were the CCR9ϪCD4ϩ T cells. Both CCR9Ϫ and CCR9ϩCD4ϩ T cells, however, proliferated when rather the result of unique activation requirements of mucosal T ϩ ϩ costimulated with anti-CD28 Abs. It has been previously shown cells (23). These data suggest that PB memory CCR9 CD4 T that mucosal T cells differ from PB-derived T cells in that the cells represent circulating mucosal T cells recently activated in former exhibit an activated phenotype yet, although proliferating vivo but not yet fully differentiated. poorly to anti-CD3 stimulation, show enhanced proliferation and ϩ ϩ cytokine secretion in response to CD2 activation (23). Because a CCR9 CD4 PB T cells exhibit a Th1 or Tr1 cytokine profile subset of circulating CCR9ϩ T cells have an activated phenotype, We have previously shown that purified small bowel CCR9ϩ and ␤ Ϫ express high levels of the mucosal homing ligand 7 integrin, and CCR9 T cells exhibit a dominant Th1 cytokine profile and that increase in frequency with small bowel inflammatory diseases, we the small percentage of Th2-producing cells were equally repre- tested the hypothesis that these cells preferentially respond to CD2 sented within the CCR9ϩ and CCR9Ϫ subset of small bowel T activation and therefore could potentially represent circulating mu- lymphocytes, suggesting that CCR9 expression is not linked to a cosal T cells. As shown in Fig. 2A, CCR9ϩCD4ϩ T cells prolif- Th1 or Th2 cytokine profile but rather to a phenotype with selec- erated vigorously with anti-CD2 activation, whereas the tive homing potential to the small bowel mucosa (21). To gain CD4ϩCCR9Ϫ cells were unresponsive. Both subsets proliferated better insight into the cytokine production profile of circulating vigorously when costimulated with CD28. The CD2 responsive- CCR9ϩCD4ϩ cells, we first examined IFN-␥, IL-2, and IL-4 pro- ness of the CCR9ϩCD4ϩ cells was present in highly purified duction at the single-cell level by intracellular cytokine staining. memory (CD45ROϩ) cells, whereas the CD45ROϩ subset of We also studied the expression of IL-10, which is produced by CCR9ϪCD4ϩ cells was unresponsive to CD2 stimulation (Fig. mucosal T cells and implicated in immunological tolerance in the 2B). Therefore, the responsiveness of CCR9ϩCD4ϩ T cells to intestine. The percentage of IFN-␥-producing cells was higher in CD2 stimulation was not simply due to the higher percentage of CCR9ϩ than in CCR9ϪCD4ϩ T cells (Fig. 3, A and C), but there memory cells contained within this cell subset compared with was no difference in the percentage of IL-4- or IL-2-producing CD4ϩCCR9Ϫ T cells. The CD2 responsiveness of mucosal T cells cells among CCR9ϩCD4ϩ and CCR9ϪCD4ϩ T cells (Fig. 3, A compared with PB T cells, shown previously by our laboratory and and B). Interestingly, IL-10-producing CD4ϩ T cells were detected others, is not simply the result of the memory T cell phenotype, but exclusively within the CCR9ϩ subset of CD4ϩ T cells (Fig. 3, B 162 CIRCULATING CCR9ϩ LYMPHOCYTES HAVE MUCOSAL T CELL CHARACTERISTICS Downloaded from

FIGURE 4. Detection of IFN-␥ and IL-10 in 24-h culture supernatant of sorted CCR9ϩ and CCR9ϪCD4ϩ PB T cells. Highly purified CCR9ϩ and CCR9Ϫ CD4ϩ T cells (105 cells/well) were stimulated with plastic-bound http://www.jimmunol.org/ FIGURE 3. Circulating CCR9ϩCD4ϩ T cells exhibit a Th1/Tr1 cyto- anti-CD3 (1 ␮g/ml) Ϯ soluble CD28 (2 ␮g/ml), or soluble anti-CD2 (1 kine phenotype. Sorted CCR9ϩ and CCR9Ϫ CD4ϩ T cells were stimulated ␮g/ml) Ϯ CD28 (2 ␮g/ml) Ab for 24 h. IFN-␥ and IL-10 were measured with PMA (50 ng/ml) and ionomycin (1 ␮g/ml) for 5 h. Brefeldin A (10 in culture supernatant by ELISA using matched paired Abs. Data are rep- ␮g/ml) was added 2 h after the initiation of culture. Intracellular staining resentative of at least five experiments from different donors. for IL-4, IFN-␥, IL-2, and IL-10 was analyzed by two-color flow cytometry. Because of the small number of sorted cells, analysis of intracellular cytokine expression was not always possible for all cytokine combinations from a single donor simultaneously. The results shown are representative Ab (Fig. 5B). CD2 Ϯ CD28 stimulation induced detectable of three experiments for each cytokine combination examined from differ- amounts of IFN-␥ or IL-10 only by the memory subset of by guest on September 28, 2021 ent donors. CCR9ϩCD4ϩ T cells (Fig. 5). The rapid production of IL-10 by CCR9ϩCD4ϩ T cells detected by intracellular staining and the detection of high levels of IL-10 in culture supernatants of memory ϩ ϩ and C). Costaining of CCR9ϩCD4ϩ T cells for IFN-␥ and -IL-10 CCR9 CD4 T cells after only 24 h show that a subset of these showed that these cytokines are produced largely by distinct cell cells rapidly produce large amounts of IL-10 early on, typical of subsets (Fig. 3C). To confirm the cytokine profile of CCR9ϩ and Tr1 cells (24). CCR9ϪCD4ϩ T cells, we examined the production of IFN-␥ and Memory CCR9ϩCD4ϩ cells provide B cell help for Ig IL-10 in culture supernatant after stimulation with anti-CD3 Ϯ production CD28 Ab. Large amounts of IFN-␥ and IL-10 were detected in the 24-h culture supernatant of CCR9ϩCD4ϩ T cells stimulated with We hypothesized that the cytokine profile and activated phenotype ϩ anti-CD3 Ϯ CD28 Ab (Fig. 4). IFN-␥ was detected at lower levels of PB CCR9 T cells, including the expression of CD40L, would in the supernatant of CCR9ϪCD4ϩ T cells, and IL-10 at much promote B cell activation, and Ig production (25, 26). Therefore, ϩ ϩ lower levels, compared with that in the supernatant of we analyzed the ability of sorted CCR9 CD4 T cells to provide CCR9ϩCD4ϩ T cells (Fig. 4). Because CCR9ϩCD4ϩ T cells pro- B cell help for Ab production in an in vitro assay (9, 10). Coculture ϩ Ϫ ϩ liferate in response to CD2 stimulation, we examined whether they experiments of memory CCR9 or CCR9 CD4 T cells with au- ϩ also produce cytokines when activated through the CD2 pathway. tologous PB CD19 B cells in the absence of added T cell stimuli ϩ ϩ As shown in Fig. 4, large amounts of IFN-␥ and IL-10 were de- or exogenous cytokines showed that CCR9 CD4 T cells are po- tected in the supernatant of CCR9ϩCD4ϩ T cells after stimulation tent inducers of IgA, IgG, and IgM production (Fig. 6). B cells Ϫ ϩ with CD2 Ϯ CD28 Ab, whereas neither cytokine was detected in cultured alone or in the presence of CCR9 CD4 T cells produced supernatant from CCR9ϪCD4ϩ T cells stimulated by CD2 Ϯ little Abs. Stimulation of B cells with PWM was the positive con- CD28 Ab. trol for Ig production (Fig. 6). We next examined IFN-␥ and IL-10 production by anti-CD3 or -CD2 stimulated memory CCR9ϩ and CCR9ϪCD4ϩ T cells, and Discussion naive CCR9ϩCD4ϩ T cells (Fig. 5). Significantly more IFN-␥ and The chemokine TECK/CCL25 and its receptor CCR9 has been IL-10 were detected in 24-h supernatant of memory CCR9ϩ than suggested to play an important role in small bowel immunity (20, in memory CCR9Ϫ or naive CCR9ϩCD4ϩ cells (Fig. 5A). Similar 21), and CCR9ϩCD4ϩ T cells may be involved in the pathogen- results were obtained when we analyzed IFN-␥ and IL-10 in 72-h esis of small bowel inflammatory diseases, because a higher frac- culture supernatant (Fig. 5B). Interestingly, IL-10 was detected at tion of CCR9ϩCD4ϩ T cells were detected in venous blood of 72 h for both memory CCR9Ϫ and naive CCR9ϩCD4ϩ cells, but patients with small intestinal Crohn’s and celiac disease, compared only when cells were stimulated with both anti-CD3 and -CD28 with patients with isolated colonic Crohn’s or normal donors (27). The Journal of Immunology 163

FIGURE 5. Memory (m) CCR9ϩCD4ϩ cells produce large amounts of IFN-␥ and IL-10. Freshly isolated PBL were incubated with 3C3/IgG2b-TC secondary Ab, -CD4- FITC, and -CD45RO-PE; sorted into CD4ϩ CD45ROϩCCR9ϩ, CD4ϩCD45ROϩCCR9Ϫ, or CD4ϩCD45ROϪCCR9ϩ; and plated in 96- well ﬂat-bottom plates coated with anti-CD3 (1 ␮g/ml) with or without anti-CD28 (2 ␮g/ml) at a concentration of 5 ϫ 104 cells/well. Under other conditions, cells were stimulated with soluble CD2 (1 ␮g/ml) with or without CD28 (2 ␮g/ml) for up to 72 h. Culture supernatants at 24 h (A) and 72 h (B) were analyzed for IFN-␥ and IL-10. Data are representative of two different donors examined. n, Naive. Downloaded from

These CCR9ϩ T cells may have been mobilized from small intes- IFN-␥ or IL-10 promptly after in vitro stimulation and, therefore,

tinal draining lymph nodes during an ongoing immune response contain differentiated Th1 and Tr1 cells. It is interesting that a http://www.jimmunol.org/ (27). Moreover, TECK/CCL25 is up-regulated in inflamed small similar cytokine profile, e.g., production of IFN-␥ and IL-10, is intestinal, but not colonic, mucosa in close proximity to areas of seen for stimulated mucosal T cells (21, 35). Therefore, it is tempt- lymphocyte infiltrates, suggesting a key role for TECK/CCL25 in ing to speculate that Th1 and Tr1 CCR9ϩCD4ϩ T cells are con- the attraction of T lymphocytes to the inflamed small intestinal tinuously generated in the inductive sites of the small intestinal mucosa in Crohn’s disease (27). Recent studies have shown that mucosal immune system and play an important role in effector and CCR9-deficient mice have fewer ␥␦TCR intraepithelial lympho- regulatory functions in the intestine (36, 37). Further studies are cytes in the small intestine but more in colon and, surprisingly, required to characterize each cell subset within CCR9ϩCD4ϩ PB normal numbers of small intestinal lamina propria lymphocytes in T cells. the small intestine and practically normal thymic T cell develop- by guest on September 28, 2021 ment (28, 29). Recent data also show that TECK/CCL25 plays an important role in the generation of murine cryptopatches and sub- sequent appearance of intraepithelial lymphocytes, localization of activated CD8␣␤ϩ lymphocytes to the small intestine, and che- moattraction of IgA-producing cells in vitro (30–32). In the current study, we analyzed the phenotype and effector function of the small subset of CCR9ϩ PB T cells from normal human donors. We hypothesized that this subset, especially the CD45ROϩCCR9ϩ cells, represent circulating, recently activated mucosal T cells homing back to the small bowel lamina propria (33, 34). This idea is supported by the observations that CCR9ϩ PB T cells 1) contain high fractions positive for activation markers and costimulatory molecules, 2) respond to CD2 as well as to CD3 stimulation (the former a signature activation pathway of mucosal T cells), 3) contain both Th1 and Tr1 cells and 4) support Ig, including IgA, production by cocultured CD19ϩ B cells without additional T cell activation or added cytokines. The most substan- tial difference in the phenotypic marker expression between CCR9ϩ and CCR9Ϫ T cells was the high fraction expressing CD25, CD69, CD71, HLA-DR, and the costimulatory molecules OX-40 and CD40L in CCR9ϩ cells, consistent with a phenotype of FIGURE 6. Memory (m) CCR9ϩCD4ϩ T cells provide B cell help in Ig activated T cells. production. Freshly isolated PBL were incubated with 3C3/IgG2b-TC sec- In addition, because CD2 responsiveness of freshly isolated T ondary Ab, -CD4-FITC, and -CD45RO-PE and sorted into CD4ϩCD45ROϩCCR9ϩ, CD4ϩCD45ROϩCCR9Ϫ cells. Sorted cells (105) cells has been documented for those cells derived from the intes- 5 ϩ Ϫ ϩ were cocultured with 10 highly purified autologous CD19 circulating B tinal mucosa and because highly purified memory CCR9 CD4 T cells in 96-well round-bottom plates for 11 days in the absence of T cell cells were unresponsive to CD2 stimulation, the CD2 responsive- ϩ ϩ stimuli or exogenous cytokines. IgM, IgA, and IgG were analyzed in cul- ness of CCR9 CD4 memory cells in blood strongly suggests that ture supernatant by ELISA. Stimulation of CD19ϩ B cells with medium they are generated in the inductive sites of the mucosal immune alone or PWM (5 ␮g/ml) was used as negative and positive control, re- system. Analysis of the effector function of circulating memory spectively. Data represent the mean value Ϯ SD of replicate T-B ϩ ϩ CCR9 CD4 T cells revealed that they produce large amounts of cocultures. 164 CIRCULATING CCR9ϩ LYMPHOCYTES HAVE MUCOSAL T CELL CHARACTERISTICS

Human and mouse Tr1 or Tr1-like cells have been generated in er’s patches and/or draining mesenteric lymph node. Because ac- vitro after manipulation of T cells with cytokines or drugs or under tivation of naive CD4ϩ T cells in vitro in the presence of several certain stimulatory conditions with APC and costimulatory mole- cytokines, including IL-10 or TGF-␤, is not sufficient to induce cules (38–43), but this is the first report to our knowledge that CCR9 (18), its expression may require contact with specialized identifies cells with the characteristics of human Tr1 cells de novo, APC in the mucosal environment (49, 50). In mice, intestinal im- ␣ ␤ within freshly isolated PB T cells of normal donors, to be restricted munization with Ag leads to up-regulation of 4 7 and the acqui- to a specific T cell subpopulation. Groux et al. (38, 44) have shown sition of TECK/CCL25 responsiveness in intestinal lymph nodes that chronic activation of both human and murine CD4ϩ T cells in by T cells (50). Therefore, the induction of CCR9 expression by T the presence of IL-10 gave rise to the development of CD4ϩ T cell cells activated in the inductive sites of the mucosal immune sys- clones with low proliferative capacity. This IL-10-mediated anergy tem, with the concomitant generation of both inflammatory (Th1) was associated with induction of a population of Tr1 cells produc- and regulatory (Tr1) T cells, would enable the small intestinal ing high levels of IL-10, low levels of IL-2, and no IL-4, which mucosal immune system to mount protective immunity to patho- could suppress Ag-specific responses in vivo and in vitro (38). gens and at the same time immune tolerance to dietary Ags and Activation of human CD4ϩ T cells with immature dendritic cells commensal bacteria. Undoubtedly, elucidation of the mecha- or anti-CD2-costimulated CD4ϩ T cells also led to the generation nism(s) of CCR9 induction in human T cells and of the require- of Tr1 cells (39, 40). Barrat et al. (41) showed that repeated stim- ments for generation of CCR9ϩ Th1 and Tr1 cells will provide ulation of human and murine naive CD4ϩ T cells in the presence important insights into human small intestinal immunity and its of dexamethasone-1,25(OH)2-vitamin D3 and anti-Th1/Th2 polar- perturbations during inflammatory diseases, and form the basis for izing cytokine Ab leads to the development of Tr1 cells as well. the development of novel therapeutics. Downloaded from Kemper et al. (45) have recently shown that coengagement of CD3 and the complement regulator CD46 in the presence of IL-2 in- Acknowledgments duces a Tr1-specific cytokine phenotype in human CD4ϩ T cells. ϩ We thank Joanne Gaiennie for providing blood from donors, Patricia Lin How could CCR9 Tr1 cells arise? Several mechanisms may op- for flow cytometry, Richard Deem for generating the figures, and Sheena erate in the small bowel mucosal immune system to induce and Lin for statistical analysis. ϩ maintain CCR9 Tr1 cells: 1) continuous exposure to dietary, http://www.jimmunol.org/ commensal bacterial or other intestine-derived Ags may lead to the References generation of Ag-specific Tr1 cells after these Ags are presented to 1. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and naive T cells in draining lymph nodes by immature dendritic cells their role in immunity. Immunity 12:121. in the absence of costimulation; 2) acquisition of CD2 responsive- 2. Baggiolini, M., and P. Loetscher. 2000. Chemokines in inflammation and immu- ness of CCR9ϩCD4ϩT cells in the mucosa could further induce nity. Immunol. Today 21:418. 3. Butcher, E. C., M. Williams, K. Youngman, L. Rott, and M. Briskin. 1999. their differentiation into Tr1 cells (40) after recruitment into the Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209. lamina propria and interaction with CD58, which is expressed by 4. Campbell, J. J., and E. C. Butcher. 2000. Chemokines in tissue-specific and mucosal B cells and intestinal epithelial cells (46). Further iden- microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12:336.

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