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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 ¹

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

^* Burns and Allen Research Institute and Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90048; Univerity Hospital of Heraklion, University of Crete, Heraklion, Crete, Greece; and Millennium Pharmaceuticals, Cambridge, MA 02139

	Abstract

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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 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 unresponsive. 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 cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.

	Introduction

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Chemokines and their receptors play a critical role in the migration of different types of leukocytes, including naive and memory/effector T lymphocytes, into microanatomic compartments of lymphoid organs and peripheral tissues (1, 2, 3, 4, 5). T cells in peripheral blood (PB)³ express a diverse set of chemokine receptors, and studies in the last few years have supported the notion that the patterns of chemokine receptors characterize functional T cell subpopulations. Naive T cells, which preferentially migrate to lymph nodes and Peyer’s patches, express CXCR4, the receptor for stromal cell-derived factor1/CXC ligand 12, and CCR7, the receptor for Epstein-Barr virus-induced molecule1 ligand chemokine/C-C chemokine ligand (CCL) 19, and secondary lymphoid organ chemokine/CCL21. Mice deficient in CCR7 or secondary lymphoid organ chemokine/CCL21 have defective homing of naive T cells to secondary lymphoid organs (6, 7). Based on the expression of CCR7, memory T cells can be further distinguished into CCR7⁺ central memory T cells with the potential to home to lymphoid organs and CCR7-effector memory T cells with the potential to migrate to inflamed tissue (8). CXCR5 expression on memory T cells defines a subset that localizes to B cell follicles and germinal centers and supports Ig production by cocultured B cells (9, 10), whereas CCR4 and CCR8 are highly expressed by human CD4⁺CD25⁺-regulatory T cells (11). Other chemokine receptors are preferentially expressed by different Th subsets; CCR3 is expressed predominantly by Th2 cells, and CCR5 or CXCR3 are expressed by Th1 cells (12, 13, 14, 15). Expression of other chemokine receptors by peripheral blood (PB) T cells may define a tissue-specific homing potential, such as CCR4 or CCR10, which are preferentially expressed by cutaneous lymphocyte Ag⁺-skin homing T cells (16, 17), and CCR9, which is expressed by ₄₇⁺-intestine-homing lymphocytes (18, 19, 20, 21). These data suggest an important role of certain chemokines and their receptors in regulating the homing of specific T cell subsets into microanatomic compartments of lymphoid organs and peripheral tissues (4).

We and others have proposed that the chemokine thymus-expressed chemokine (TECK)/CCL25 and its receptor CCR9 may play an important role in the regional specialization of intestinal immunity and that the combined expression of CCR9 and ₄₇ on the cell surface may provide a small intestinal address code for circulating intestinal memory T cells (20, 21). CCR9 is expressed on a small subset of PB CD4⁺ (2–4%) and CD8⁺ T cells, most of which coexpress the mucosal homing ligand ₄₇ (18). Here, we show that circulating CCR9⁺CD4⁺ T cells from normal donors have characteristics of mucosal T cells in terms of an activated phenotype, proliferative response to anti-CD2 stimulation, a Th1 or T-regulatory 1 (Tr1) cytokine profile, and support for Ig production by cocultured B cells. This T cell subset may provide a peripheral window to small intestinal immunity in humans and a tool to study Ag specificity and cytokine profile of effector and regulatory cells in small bowel inflammatory diseases and in oral tolerance.

	Materials and Methods

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Abs and reagents

Anti-CD3, -CD4, -CD8, -HLA-DR dye-linked mAbs for immunofluorescence studies were obtained from Caltag (Burlingame, CA). Anti-CD25, -CD69, -₇ integrin, -CD40L, -OX-40, -CTLA, -CD27, -CD71, -CD62L, and -CD45RO dye-linked mAbs were obtained from BD PharMingen (San 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-IL-2 Ab was from Caltag. PWM was from Sigma-Aldrich (St. Louis, MO). The anti-CCR9 mAb, 3C3, was from Millennium Pharmaceuticals (Cambridge, MA).

Cell isolation, sorting, and FACS analysis

PBLs were isolated from normal healthy volunteers by separation on Ficoll-Hypaque gradients. The cells were subsequently washed three times with HBSS and resuspended in RPMI 1640 containing 2 mM L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 50 µg/ml penicillin-streptomycin, and 10% heat-inactivated FCS. The cells were stained with anti-CCR9 mAb (3C3, IgG2b) followed by a secondary PE- or tricolor (TC)-conjugated goat anti-mouse IgG2b mAb (Caltag). The cells were washed and incubated with mouse IgG for 15 min and subsequently stained with FITC-conjugated anti-CD4 mAb. After staining, the cells were sorted using FACSVantage (BD Biosciences, San Jose, CA) to isolate CD4⁺CCR9⁺ and CD4⁺CCR9^- cells (purity, >99%). In some experiments, cells were stained with anti-CCR9/TC-conjugated goat anti-mouse IgG2b mAb, PE-conjugated anti-CD45RO, and FITC-conjugated anti-CD4 mAb and sorted into CD4⁺CD45RO⁺CCR9⁺, CD4⁺CD45RO^-CCR9⁺, and CD4⁺CD45RO⁺CCR9^- cells (purity, >98%).

For staining of cell surface Ags, 5 x 10 ⁵ freshly isolated PBL were washed twice with PBS supplemented with 0.1% BSA and 0.1% azide and resuspended in 100 µl of 10% human Ab serum to block nonspecific Fc 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. After two washings, cells were resuspended in 400 µl of 1% paraformaldehyde in PBS and analyzed by FACS (BD Biosciences, San Jose, CA); 3 x 10⁴ events were routinely collected and analyzed using CellQuest software (BD Biosciences).

Cytokine detection

Sorted CD4⁺CCR9⁺ and CD4⁺CCR9^- T cells or CD4⁺CD45RO⁺CCR9⁺, CD4⁺CD45RO^-CCR9⁺, and CD4⁺CD45RO⁺CCR9^- cells were stimulated with plate-bound anti-CD3 (OKT3, 1 µg/ml; American Type Culture Collection, Manassas, VA) with or without soluble anti-CD28 (clone 9.3, 2 µg/ml) or with soluble anti-CD2 Abs (clones CB6 and GD10 at 1 µg/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 CD4⁺ T cell subsets were stimulated with 50 ng/ml PMA (Sigma) and 1 µg/ml ionomycin for 5 h. Brefeldin A (10 µg/ml) was added to the culture after 2 h of stimulation to block cytokine secretion. Cells were fixed and permeabilized using Cytofix-Cytoperm solution (Caltag) and stained with PE- and FITC-conjugated control Abs or mAbs to IL-2, IFN-, IL-4, and IL-10.

Proliferation assays

Sorted CD4⁺CCR9⁺ and CD4⁺CCR9^- T cells or CD4⁺CD45RO⁺CCR9⁺ and CD4⁺CD45RO⁺CCR9^- cells were stimulated with plate-bound anti-CD3 (1 µg/ml) with or without soluble anti-CD28 (2 µg/ml) or with soluble anti-CD2 (1 µg/ml) with or without anti-CD28 and pulsed with 1 µCi/well [³H]thymidine for the last 16 h of a 96-h culture. Radioactivity was analyzed on a scintillation counter.

Ab production

PB T cells (CD4⁺CD45RO⁺CCR9⁺ or CD4⁺CD45RO⁺CCR9^-) and B cells (CD19⁺) were sorted by FACS and cocultured in round-bottom 96-well plates at 10⁵ cells/well each of T and B cells in the absence of T cell activation stimuli or cytokines for 11 days. B cells stimulated with PWM (5 µg/ml) were used as positive control. IgM, IgG, and IgA contents in the culture supernatants were determined by ELISA.

Statistical analysis

Differences between the percentage of phenotypic markers expressed between CCR9⁺ and CCR9^- T cells were compared with a paired t test. p < 0.05 was considered statistically significant.

	Results

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CCR9⁺ PB T cells contain a higher fraction expressing surface activation and costimulatory molecules than does the CCR9^- subset

We have recently shown that patients with small intestinal inflammatory diseases, such as Crohn’s and celiac disease, have a higher percentage of circulating CCR9⁺CD4⁺ T cells than do patients with colonic Crohn’s or normal healthy donors; therefore, this cell subset is potentially involved in these small intestinal inflammatory processes. To gain insight into the functional aspects of the CCR9⁺ PB T cells from normal donors, we first examined the expression of several activation markers and costimulatory molecules on CCR9⁺ PB T cells. As shown in Fig. 1 and Table I, more CCR9⁺ T cells express CD4 and CD8 and the activation markers CD25, CD69, CD71, and HLA-DR than do CCR9^- T cells. Similarly, the fraction of cells expressing OX-40 and CD40L was higher in CCR9⁺ than in CCR9^- T cells. The CD27⁺ fraction was similar in the two cell subsets. Most CCR9⁺ and CCR9^- T cells coexpress CD62L, but the positive fraction was lower for circulating CCR9⁺ compared with CCR9^- T cells. Additionally, more CCR9⁺ T cells were CD45RO⁺ and ₇ integrin⁺ as compared with the CCR9^- T cells, as previously described (see also Fig. 1 and Table I). Collectively, our data show that a subset of circulating CCR9⁺ T cells have a phenotype associated with activation. Addition of CD4⁺ and CD8⁺ percentages of CCR9⁺ T cells gives >100% (see Fig. 1 and Table I), indicating that a subset of CCR9⁺ T cells must be double-positive T cells, which is consistent with their activation phenotype (22).

View larger version (59K): [in this window] [in a new window]   FIGURE 1. Expression of activation and costimulatory molecules in freshly isolated PB CCR9+ T lymphocytes. Freshly isolated PBL from normal donors were stained with 3C3/IgG2b-TC secondary Ab, anti-CD3, and Ab against several activation and costimulatory molecules and analyzed by FACS. The cells were gated on CD3+ lymphocytes. The percentages in the quadrants represent the percent of CCR9+ (upper right) or CCR9- (lower right) T lymphocytes that express each cell surface marker shown in each dot plot. Representative data of three to five different donors examined.

To determine the activation requirement of PB CCR9⁺CD4⁺ T cells, sorted CD4⁺CCR9⁺ and CD4⁺CCR9^- T cells were stimulated with IL-2, or anti-CD3 ± CD28, or anti-CD2 ± CD28, and proliferation of each cell subset was analyzed. As shown in Fig. 2A, CCR9⁺CD4⁺T cells were more responsive to IL-2 or anti-CD3 stimulation alone than were the CCR9^-CD4⁺ T cells. Both CCR9^- and CCR9⁺CD4⁺ T cells, however, proliferated when costimulated with anti-CD28 Abs. It has been previously shown that mucosal T cells differ from PB-derived T cells in that the former exhibit an activated phenotype yet, although proliferating poorly to anti-CD3 stimulation, show enhanced proliferation and cytokine secretion in response to CD2 activation (23). Because a subset of circulating CCR9⁺ T cells have an activated phenotype, express high levels of the mucosal homing ligand ₇ integrin, and increase in frequency with small bowel inflammatory diseases, we tested the hypothesis that these cells preferentially respond to CD2 activation and therefore could potentially represent circulating mucosal T cells. As shown in Fig. 2A, CCR9⁺CD4⁺ T cells proliferated vigorously with anti-CD2 activation, whereas the CD4⁺CCR9^- cells were unresponsive. Both subsets proliferated vigorously when costimulated with CD28. The CD2 responsiveness of the CCR9⁺CD4⁺ cells was present in highly purified memory (CD45RO⁺) cells, whereas the CD45RO⁺ subset of CCR9^-CD4⁺ cells was unresponsive to CD2 stimulation (Fig. 2B). Therefore, the responsiveness of CCR9⁺CD4⁺ T cells to CD2 stimulation was not simply due to the higher percentage of memory cells contained within this cell subset compared with CD4⁺CCR9^- T cells. The CD2 responsiveness of mucosal T cells compared with PB T cells, shown previously by our laboratory and others, is not simply the result of the memory T cell phenotype, but rather the result of unique activation requirements of mucosal T cells (23). These data suggest that PB memory CCR9⁺CD4⁺ T cells represent circulating mucosal T cells recently activated in vivo but not yet fully differentiated.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Proliferative responses of sorted CCR9+and CCR9- CD4+ T cells to IL-2, anti-CD3, or anti-CD2 stimulation. A, PBL were stained with 3C3/IgG2b-PE secondary Ab and CD4 Abs and sorted into two cell subsets, CD4+CCR9+ and CD4+CCR9-. Sorted cells (105/wells) were plated in flat-bottom 96-well plates coated with anti-CD3 (OKT3, 1 µg/ml). Under other conditions, cells were incubated with IL-2 (200 U/ml), anti-CD2 (1 µg/ml), and anti-CD28 (2 µg/ml). Proliferation was determined at day 4, with [3H]thymidine added for the last 16 h of culture. B, Proliferative responses of memory (m) CCR9+ and CCR9-CD4+ T cells to anti-CD3 or anti-CD2 stimulation. Cells were incubated with 3C3/IgG2b-TC secondary Ab, -CD4-FITC, and -CD45RO-PE; sorted into CD4+CD45RO+CCR9+, CD4+CD45RO+CCR9-; and plated at a concentration of 5 x 104 cells/well. Proliferation was determined at day 4, with [3H]thymidine added for the last 16 h of culture.

We have previously shown that purified small bowel CCR9⁺ and CCR9^- T cells exhibit a dominant Th1 cytokine profile and that the small percentage of Th2-producing cells were equally represented within the CCR9⁺ and CCR9^- subset of small bowel T lymphocytes, suggesting that CCR9 expression is not linked to a Th1 or Th2 cytokine profile but rather to a phenotype with selective homing potential to the small bowel mucosa (21). To gain better insight into the cytokine production profile of circulating CCR9⁺CD4⁺ cells, we first examined IFN-, IL-2, and IL-4 production at the single-cell level by intracellular cytokine staining. We also studied the expression of IL-10, which is produced by mucosal T cells and implicated in immunological tolerance in the intestine. The percentage of IFN--producing cells was higher in CCR9⁺ than in CCR9^-CD4⁺ T cells (Fig. 3, A and C), but there was no difference in the percentage of IL-4- or IL-2-producing cells among CCR9⁺CD4⁺ and CCR9^-CD4⁺ T cells (Fig. 3, A and B). Interestingly, IL-10-producing CD4⁺ T cells were detected exclusively within the CCR9⁺ subset of CD4⁺ T cells (Fig. 3, B and C). Costaining of CCR9⁺CD4⁺ T cells for IFN- and -IL-10 showed that these cytokines are produced largely by distinct cell subsets (Fig. 3C). To confirm the cytokine profile of CCR9⁺ and CCR9^-CD4⁺ T cells, we examined the production of IFN- and IL-10 in culture supernatant after stimulation with anti-CD3 ± CD28 Ab. Large amounts of IFN- and IL-10 were detected in the 24-h culture supernatant of CCR9⁺CD4⁺ T cells stimulated with anti-CD3 ± CD28 Ab (Fig. 4). IFN- was detected at lower levels in the supernatant of CCR9^-CD4⁺ T cells, and IL-10 at much lower levels, compared with that in the supernatant of CCR9⁺CD4⁺ T cells (Fig. 4). Because CCR9⁺CD4⁺ T cells proliferate in response to CD2 stimulation, we examined whether they also produce cytokines when activated through the CD2 pathway. As shown in Fig. 4, large amounts of IFN- and IL-10 were detected in the supernatant of CCR9⁺CD4⁺ T cells after stimulation with CD2 ± CD28 Ab, whereas neither cytokine was detected in supernatant from CCR9^-CD4⁺ T cells stimulated by CD2 ± CD28 Ab.

View larger version (51K): [in this window] [in a new window]   FIGURE 3. Circulating CCR9+CD4+ T cells exhibit a Th1/Tr1 cytokine phenotype. Sorted CCR9+ and CCR9- CD4+ T cells were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) for 5 h. Brefeldin A (10 µg/ml) was added 2 h after the initiation of culture. Intracellular staining 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 of three experiments for each cytokine combination examined from different donors.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Detection of IFN- and IL-10 in 24-h culture supernatant of sorted CCR9+ and CCR9-CD4+ PB T cells. Highly purified CCR9+ and CCR9- CD4+ T cells (105 cells/well) were stimulated with plastic-bound anti-CD3 (1 µg/ml) ± soluble CD28 (2 µg/ml), or soluble anti-CD2 (1 µg/ml) ± CD28 (2 µg/ml) Ab for 24 h. IFN- and IL-10 were measured in culture supernatant by ELISA using matched paired Abs. Data are representative of at least five experiments from different donors.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Memory (m) CCR9+CD4+ 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 CD4+CD45RO+CCR9+, CD4+CD45RO+CCR9-, or CD4+CD45RO-CCR9+; and plated in 96-well flat-bottom plates coated with anti-CD3 (1 µg/ml) with or without anti-CD28 (2 µg/ml) at a concentration of 5 x 104 cells/well. Under other conditions, cells were stimulated with soluble CD2 (1 µg/ml) with or without CD28 (2 µg/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.

We hypothesized that the cytokine profile and activated phenotype of PB CCR9⁺ T cells, including the expression of CD40L, would promote B cell activation, and Ig production (25, 26). Therefore, we analyzed the ability of sorted CCR9⁺CD4⁺ T cells to provide B cell help for Ab production in an in vitro assay (9, 10). Coculture experiments of memory CCR9⁺ or CCR9^-CD4⁺ T cells with autologous PB CD19⁺ B cells in the absence of added T cell stimuli or exogenous cytokines showed that CCR9⁺CD4⁺ T cells are potent inducers of IgA, IgG, and IgM production (Fig. 6). B cells cultured alone or in the presence of CCR9^-CD4⁺ T cells produced little Abs. Stimulation of B cells with PWM was the positive control for Ig production (Fig. 6).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Memory (m) CCR9+CD4+ T cells provide B cell help in Ig production. Freshly isolated PBL were incubated with 3C3/IgG2b-TC secondary Ab, -CD4-FITC, and -CD45RO-PE and sorted into CD4+CD45RO+CCR9+, CD4+CD45RO+CCR9- cells. Sorted cells (105) were cocultured with 105 highly purified autologous CD19+ circulating B cells in 96-well round-bottom plates for 11 days in the absence of T cell stimuli or exogenous cytokines. IgM, IgA, and IgG were analyzed in culture supernatant by ELISA. Stimulation of CD19+ B cells with medium alone or PWM (5 µg/ml) was used as negative and positive control, respectively. Data represent the mean value ± SD of replicate T-B cocultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the current study, we analyzed the phenotype and effector function of the small subset of CCR9⁺ PB T cells from normal human donors. We hypothesized that this subset, especially the CD45RO⁺CCR9⁺ 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 CCR9⁺ 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 CD19⁺ B cells without additional T cell activation or added cytokines. The most substantial difference in the phenotypic marker expression between CCR9⁺ and CCR9^- T cells was the high fraction expressing CD25, CD69, CD71, HLA-DR, and the costimulatory molecules OX-40 and CD40L in CCR9⁺ cells, consistent with a phenotype of activated T cells.

In addition, because CD2 responsiveness of freshly isolated T cells has been documented for those cells derived from the intestinal mucosa and because highly purified memory CCR9^-CD4⁺ T cells were unresponsive to CD2 stimulation, the CD2 responsiveness of CCR9⁺CD4⁺ memory cells in blood strongly suggests that they are generated in the inductive sites of the mucosal immune system. Analysis of the effector function of circulating memory CCR9⁺CD4⁺ T cells revealed that they produce large amounts of IFN- or IL-10 promptly after in vitro stimulation and, therefore, contain differentiated Th1 and Tr1 cells. It is interesting that a similar cytokine profile, e.g., production of IFN- and IL-10, is seen for stimulated mucosal T cells (21, 35). Therefore, it is tempting to speculate that Th1 and Tr1 CCR9⁺CD4⁺ T cells are continuously generated in the inductive sites of the small intestinal mucosal immune system and play an important role in effector and regulatory functions in the intestine (36, 37). Further studies are required to characterize each cell subset within CCR9⁺CD4⁺ PB T cells.

Human and mouse Tr1 or Tr1-like cells have been generated in vitro after manipulation of T cells with cytokines or drugs or under certain stimulatory conditions with APC and costimulatory molecules (38, 39, 40, 41, 42, 43), but this is the first report to our knowledge that identifies cells with the characteristics of human Tr1 cells de novo, within freshly isolated PB T cells of normal donors, to be restricted to a specific T cell subpopulation. Groux et al. (38, 44) have shown that chronic activation of both human and murine CD4⁺ T cells in the presence of IL-10 gave rise to the development of CD4⁺ T cell clones with low proliferative capacity. This IL-10-mediated anergy was associated with induction of a population of Tr1 cells producing high levels of IL-10, low levels of IL-2, and no IL-4, which could suppress Ag-specific responses in vivo and in vitro (38). Activation of human CD4⁺ T cells with immature dendritic cells or anti-CD2-costimulated CD4⁺ T cells also led to the generation of Tr1 cells (39, 40). Barrat et al. (41) showed that repeated stimulation of human and murine naive CD4⁺ T cells in the presence of dexamethasone-1,25(OH)₂-vitamin D₃ and anti-Th1/Th2 polarizing cytokine Ab leads to the development of Tr1 cells as well. Kemper et al. (45) have recently shown that coengagement of CD3 and the complement regulator CD46 in the presence of IL-2 induces a Tr1-specific cytokine phenotype in human CD4⁺ T cells. How could CCR9⁺ Tr1 cells arise? Several mechanisms may operate in the small bowel mucosal immune system to induce and maintain CCR9⁺ Tr1 cells: 1) continuous exposure to dietary, commensal bacterial or other intestine-derived Ags may lead to the generation of Ag-specific Tr1 cells after these Ags are presented to naive T cells in draining lymph nodes by immature dendritic cells in the absence of costimulation; 2) acquisition of CD2 responsiveness of CCR9⁺CD4⁺T cells in the mucosa could further induce their differentiation into Tr1 cells (40) after recruitment into the lamina propria and interaction with CD58, which is expressed by mucosal B cells and intestinal epithelial cells (46). Further identification of the surface phenotype of CCR9⁺CD4⁺ IL-10-producing T cells from PB could provide important insight into the mechanism of generation of this T cell subset in humans and lead to new strategies for immunotherapy in small bowel inflammatory disease, such as Crohn’s disease.

One important functional aspect of memory CCR9⁺CD4⁺ T cells is their ability to support Ab production by cocultured B cells, including IgA, further supporting their mucosal origin. B cell helper activity for Ab production has been reported for tonsillar but not PB CXCR5⁺ T cells (9, 10, 47). The support for Ab production by cocultured B cells provided by memory CCR9⁺CD4⁺ T cells without added T cell stimulation is consistent with their activated phenotype, particularly their expression of CD40L and their ability to produce IL-10. We have detected, in some experiments, significant levels of IL-10 in 24-h culture supernatants of sorted memory CCR9⁺CD4⁺ T cells with no added T cell-activating agents (data not shown), suggesting that these cells have been recently activated in vivo before their isolation. We have not yet tested whether memory CCR9⁺CD4⁺ T cells simply stimulate Ig production by differentiated B cells, or induce differentiation and isotype switching by naive B cells, or both. Another explanation for the B cell helper activity of circulating memory CCR9⁺CD4⁺ T cells may lie in their responsiveness to CD2 signaling. B cells could further activate these T cells during culture through LFA-3/CD2 or LFA-1/ICAM-1 interactions (48), leading to up-regulation of CD40L expression, cytokine secretion, and efficient help for Ig production. Further studies are needed to test these possibilities.

Our data provide strong evidence that human PB memory CCR9⁺CD4⁺ T cells represent circulating intestinal mucosal T cells, which have been recently activated in vivo, possibly in Peyer’s patches and/or draining mesenteric lymph node. Because activation of naive CD4⁺ T cells in vitro in the presence of several cytokines, including IL-10 or TGF-, is not sufficient to induce CCR9 (18), its expression may require contact with specialized APC in the mucosal environment (49, 50). In mice, intestinal immunization with Ag leads to up-regulation of ₄₇ and the acquisition of TECK/CCL25 responsiveness in intestinal lymph nodes by T cells (50). Therefore, the induction of CCR9 expression by T cells activated in the inductive sites of the mucosal immune system, with the concomitant generation of both inflammatory (Th1) and regulatory (Tr1) T cells, would enable the small intestinal mucosal immune system to mount protective immunity to pathogens and at the same time immune tolerance to dietary Ags and commensal bacteria. Undoubtedly, elucidation of the mechanism(s) of CCR9 induction in human T cells and of the requirements for generation of CCR9⁺ Th1 and Tr1 cells will provide important insights into human small intestinal immunity and its perturbations during inflammatory diseases, and form the basis for the development of novel therapeutics.

	Acknowledgments

 
We thank Joanne Gaiennie for providing blood from donors, Patricia Lin for flow cytometry, Richard Deem for generating the figures, and Sheena Lin for statistical analysis.

	Footnotes

 
¹ This work is supported by Grants DK-46763 and DK-56328 from the National Institutes of Health (to S.R.T.) and by a Career Development Award from the Crohn’s and Colitis Foundation of America (to K.A.P.).

² Address correspondence and reprint requests to Dr. Konstantinos A. Papadakis, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, D-4063, Los Angeles, CA 90048. E-mail address: Papadakisk{at}cshs.org or Dr. Stephan R. Targan, 8700 Beverly Boulevard, D-4063, Los Angeles, CA 90048. E-mail address: Targans{at}cshs.org

³ Abbreviations used in this paper: PB, peripheral blood; CCL, C-C chemokine ligand; TECK, thymus-expressed chemokine; Tr1, T-regulatory 1; TC, tricolor.

Received for publication December 20, 2002. Accepted for publication April 18, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Zlotnik, A., O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12:121.[Medline]
2. Baggiolini, M., P. Loetscher. 2000. Chemokines in inflammation and immunity. Immunol. Today 21:418.[Medline]
3. Butcher, E. C., M. Williams, K. Youngman, L. Rott, M. Briskin. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209.[Medline]
4. Campbell, J. J., E. C. Butcher. 2000. Chemokines in tissue-specific and microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12:336.[Medline]
5. Luster, A. D.. 1998. Chemokines: chemotactic cytokines that mediate inflammation. N. Engl. J Med. 338:436.[Free Full Text]
6. Forster, R., A. Schubel, D. Breitfeld, E. Kremmer, I. Renner-Muller, E. Wolf, M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23.[Medline]
7. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J. Exp. Med. 189:451.[Abstract/Free Full Text]
8. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708.[Medline]
9. Breitfeld, D., L. Ohl, E. Kremmer, J. Ellwart, F. Sallusto, M. Lipp, R. Forster. 2000. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J. Exp. Med. 192:1545.[Abstract/Free Full Text]
10. Schaerli, P., K. Willimann, A. B. Lang, M. Lipp, P. Loetscher, B. Moser. 2000. CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J. Exp. Med. 192:1553.[Abstract/Free Full Text]
11. Iellem, A., M. Mariani, R. Lang, H. Recalde, P. Panina-Bordignon, F. Sinigaglia, D. D’Ambrosio. 2001. Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4⁺CD25⁺ regulatory T cells. J. Exp. Med. 194:847.[Abstract/Free Full Text]
12. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005.[Abstract/Free Full Text]
13. Sallusto, F., D. Lenig, C. R. Mackay, A. Lanzavecchia. 1998. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187:875.[Abstract/Free Full Text]
14. Kim, C. H., L. Rott, E. J. Kunkel, M. C. Genovese, D. P. Andrew, L. Wu, E. C. Butcher. 2001. Rules of chemokine receptor association with T cell polarization in vivo. J. Clin. Invest. 108:1331.[Medline]
15. Loetscher, P., M. Uguccioni, L. Bordoli, M. Baggiolini, B. Moser, C. Chizzolini, J. M. Dayer. 1998. CCR5 is characteristic of Th1 lymphocytes. Nature 391:344.[Medline]
16. Campbell, J. J., G. Haraldsen, J. Pan, J. Rottman, S. Qin, P. Ponath, D. P. Andrew, R. Warnke, N. Ruffing, N. Kassam, L. Wu, E. C. Butcher. 1999. The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells. Nature 400:776.[Medline]
17. Hudak, S., M. Hagen, Y. Liu, D. Catron, E. Oldham, L. M. McEvoy, E. P. Bowman. 2002. Immune surveillance and effector functions of CCR10⁺ skin homing T cells. J. Immunol. 169:1189.[Abstract/Free Full Text]
18. Zabel, B. A., W. W. Agace, J. J. Campbell, H. M. Heath, D. Parent, A. I. Roberts, E. C. Ebert, N. Kassam, S. Qin, M. Zovko, et al 1999. Human G protein-coupled receptor GPR-9–6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis. J. Exp. Med. 190:1241.[Abstract/Free Full Text]
19. Wurbel, M. A., J. M. Philippe, C. Nguyen, G. Victorero, T. Freeman, P. Wooding, A. Miazek, M. G. Mattei, M. Malissen, B. R. Jordan, et al 2000. The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9. Eur. J. Immunol. 30:262.[Medline]
20. Kunkel, E. J., J. J. Campbell, G. Haraldsen, J. Pan, J. Boisvert, A. I. Roberts, E. C. Ebert, M. A. Vierra, S. B. Goodman, M. C. Genovese, et al 2000. Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity. J. Exp. Med. 192:761.[Abstract/Free Full Text]
21. Papadakis, K. A., J. Prehn, V. Nelson, L. Cheng, S. W. Binder, P. D. Ponath, D. P. Andrew, S. R. Targan. 2000. The role of thymus-expressed chemokine and its receptor CCR9 on lymphocytes in the regional specialization of the mucosal immune system. J. Immunol. 165:5069.[Abstract/Free Full Text]
22. Blue, M. L., J. F. Daley, H. Levine, S. F. Schlossman. 1985. Coexpression of T4 and T8 on peripheral blood T cells demonstrated by two-color fluorescence flow cytometry. J. Immunol. 134:2281.[Abstract]
23. Targan, S. R., R. L. Deem, M. Liu, S. Wang, A. Nel. 1995. Definition of a lamina propria T cell responsive state: enhanced cytokine responsiveness of T cells stimulated through the CD2 pathway. J. Immunol. 154:664.[Abstract]
24. Levings, M. K., M. G. Roncarolo. 2000. T-regulatory 1 cells: a novel subset of CD4 T cells with immunoregulatory properties. J. Allergy Clin. Immunol. 106:S109.[Medline]
25. Armitage, R. J., B. M. Macduff, M. K. Spriggs, W. C. Fanslow. 1993. Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand are modulated by soluble cytokines. J. Immunol. 150:3671.[Abstract]
26. Zan, H., A. Cerutti, P. Dramitinos, A. Schaffer, P. Casali. 1998. CD40 engagement triggers switching to IgA1 and IgA2 in human B cells through induction of endogenous TGF-: evidence for TGF- but not IL-10-dependent direct SµS and sequential SµS, SS DNA recombination. J. Immunol. 161:5217.[Abstract/Free Full Text]
27. Papadakis, K. A., J. Prehn, S. T. Moreno, L. Cheng, E. A. Kouroumalis, R. Deem, T. Breaverman, P. D. Ponath, D. P. Andrew, P. H. Green, et al 2001. CCR9-positive lymphocytes and thymus-expressed chemokine distinguish small bowel from colonic Crohn’s disease. Gastroenterology 121:246.[Medline]
28. Wurbel, M. A., M. Malissen, D. Guy-Grand, E. Meffre, M. C. Nussenzweig, M. Richelme, A. Carrier, B. Malissen. 2001. Mice lacking the CCR9 CC-chemokine receptor show a mild impairment of early T- and B-cell development and a reduction in T-cell receptor ⁺ gut intraepithelial lymphocytes. Blood 98:2626.[Abstract/Free Full Text]
29. Uehara, S., A. Grinberg, J. M. Farber, P. E. Love. 2002. A role for CCR9 in lymphocyte development and migration. J. Immunol. 168:2811.[Abstract/Free Full Text]
30. Onai, N., M. Kitabatake, Y. Y. Zhang, H. Ishikawa, K. Matsushima. 2002. Pivotal role of CCL25 (TECK)-CCR9 in the formation of gut cryptopatches and consequent appearance of intestinal intraepithelial T lymphocytes. Int. Immunol. 14:687.[Abstract/Free Full Text]
31. Svensson, M., J. Marsal, A. Ericsson, L. Carramolina, T. Broden, G. Marquez, W. W. Agace. 2002. CCCL25 mediates the localization of recently activated CD8⁺ lymphocytes to the small-intestinal mucosa. J. Clin. Invest. 110:1113.[Medline]
32. Bowman, E. P., N. A. Kuklin, K. R. Youngman, N. H. Lazarus, E. J. Kunkel, J. Pan, H. B. Greenberg, E. C. Butcher. 2002. The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. J. Exp. Med. 195:269.[Abstract/Free Full Text]
33. Kantele, A., J. Zivny, M. Hakkinen, C. O. Elson, J. Mestecky. 1999. Differential homing commitments of antigen-specific T cells after oral or parenteral immunization in humans. J. Immunol. 162:5173.[Abstract/Free Full Text]
34. Lee, H. O., C. J. Cooper, J. H. Choi, Z. Alnadjim, T. A. Barrett. 2002. The state of CD4⁺ T cell activation is a major factor for determining the kinetics and location of T cell responses to oral antigen. J. Immunol. 168:3833.[Abstract/Free Full Text]
35. Braunstein, J., L. Qiao, F. Autschbach, G. Schurmann, S. Meuer. 1997. T cells of the human intestinal lamina propria are high producers of interleukin-10. Gut 41:215.[Abstract/Free Full Text]
36. Fowler, S., F. Powrie. 2002. CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance. Eur. J. Immunol. 32:2997.[Medline]
37. Nagata, S., C. McKenzie, S. L. Pender, M. Bajaj-Elliott, P. D. Fairclough, J. A. Walker-Smith, G. Monteleone, T. T. MacDonald. 2000. Human Peyer’s patch T cells are sensitized to dietary antigen and display a Th cell type 1 cytokine profile. J. Immunol. 165:5315.[Abstract/Free Full Text]
38. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4⁺ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
39. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, A. H. Enk. 2000. Induction of interleukin 10-producing, nonproliferating CD4⁺ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192:1213.[Abstract/Free Full Text]
40. Wakkach, A., F. Cottrez, H. Groux. 2001. Differentiation of regulatory T cells 1 is induced by CD2 costimulation. J. Immunol. 167:3107.[Abstract/Free Full Text]
41. Barrat, F. J., D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul, R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, A. O’Garra. 2002. In vitro generation of interleukin 10-producing regulatory CD4⁺ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J. Exp. Med. 195:603.[Abstract/Free Full Text]
42. Gilliet, M., Y. J. Liu. 2002. Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells. J. Exp. Med. 195:695.[Abstract/Free Full Text]
43. Levings, M. K., R. Sangregorio, F. Galbiati, S. Squadrone, R. de Waal Malefyt, M. G. Roncarolo. 2001. IFN- and IL-10 induce the differentiation of human type 1 T regulatory cells. J. Immunol. 166:5530.[Abstract/Free Full Text]
44. Groux, H., M. Bigler, J. E. de Vries, M. G. Roncarolo. 1996. Interleukin-10 induces a long-term antigen-specific anergic state in human CD4⁺ T cells. J. Exp. Med. 184:19.[Abstract/Free Full Text]
45. Kemper, C., A. C. Chan, J. M. Green, K. A. Brett, K. M. Murphy, J. P. Atkinson. 2003. Activation of human CD4⁺ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421:388.[Medline]
46. Framson, P. E., D. H. Cho, L. Y. Lee, R. M. Hershberg. 1999. Polarized expression and function of the costimulatory molecule CD58 on human intestinal epithelial cells. Gastroenterology 116:1054.[Medline]
47. Kim, C. H., L. S. Rott, I. Clark-Lewis, D. J. Campbell, L. Wu, E. C. Butcher. 2001. Subspecialization of CXCR5⁺ T cells: B helper activity is focused in a germinal center-localized subset of CXCR5⁺ T cells. J. Exp. Med. 193:1373.[Abstract/Free Full Text]
48. Hirokawa, M., J. D. Gray, T. Takahashi, D. A. Horwitz. 1992. Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation. J. Immunol. 149:1859.[Abstract]
49. Campbell, D. J., E. C. Butcher. 2002. Intestinal attraction: CCL25 functions in effector lymphocyte recruitment to the small intestine. J. Clin. Invest. 110:1079.[Medline]
50. Campbell, D. J., E. C. Butcher. 2002. Rapid acquisition of tissue-specific homing phenotypes by CD4⁺ T cells activated in cutaneous or mucosal lymphoid tissues. J. Exp. Med. 195:135.[Abstract/Free Full Text]

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