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The Journal of Immunology, 2001, 166: 103-111.
Copyright © 2001 by The American Association of Immunologists

C-C Chemokine Receptor 4 Expression Defines a Major Subset of Circulating Nonintestinal Memory T Cells of Both Th1 and Th2 Potential

David P. Andrew1,*, Nancy Ruffing1,*, Chang H. Kim{dagger}, Wenyan Miao*, Heidi Heath*, You Li*, Kristine Murphy*, James J. Campbell{dagger}, Eugene C. Butcher{dagger} and Lijun Wu3,*

* Millennium Pharmaceuticals, Cambridge, MA 02139; and {dagger} Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4+ thymocytes and on a major fraction of blood nonintestinal ({alpha}4{beta}7-) memory CD4 lymphocytes, including almost all skin memory CD4+ cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4+ memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4+ CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4+ memory CD4 lymphocytes (including both CLA+ and CLA- fractions) readily express the Th1 cytokine IFN-{gamma} after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemokines are a large and growing family of 6- to 14-kDa heparin-binding proteins that mediate a wide range of biological functions (1, 2, 3, 4). In various in vitro assays, chemokines support the chemotaxis or transendothelial migration of leukocytes, while in vivo injection (5) or overexpression of chemokines (6) results in leukocyte accumulation at the site of chemokine expression. Antagonists of chemokines and their signaling prevent leukocyte trafficking (7) and have beneficial effects in several acute and chronic inflammatory models (8, 9). Chemokines also modulate angiogenesis (10) and hemopoiesis (11) as well as T lymphocyte activation (12, 13), and several act as coreceptors with CD4 for entry of M-tropic and T-tropic HIV-1 (2).

Several subsets of CD4 lymphocytes can be defined based on their expression of adhesion molecules CD62L, cutaneous lymphocyte Ag (CLA),4 {alpha}4{beta}7, and {alpha}4{beta}1, and these various CD4 lymphocyte subsets traffick to different physiologic sites (14). For example, CLA+ CD4+ embody memory for cutaneous Ags and are thought to traffic to the skin (15), while the opposite subset of {alpha}4{beta}7+ memory CD4 lymphocytes traffics to intestinal sites (16). Leukocyte adhesion to endothelium involves several overlapping steps of rolling, activation, and arrest. Exposure of rolling leukocytes to factors expressed at the adhesion site activates leukocytes and up-regulates integrin-mediated adhesion. As a result of this interaction, leukocytes arrest on the endothelium (7, 17). Leukocyte activation and up-regulation of integrin molecules occur via a pertussis toxin-sensitive mechanism that is thought to involve chemokine receptors (7, 18). A growing body of evidence indicates that leukocyte subsets will also be defined by the expression of chemokine receptors required to traffic to various physiologic sites (19).

Several chemokine receptors are expressed on subsets of memory CD4 lymphocytes. For example, CXC chemokine receptor 3 (CXCR3), CCR2, and CCR5 (20, 21, 22) are all expressed on subsets of memory CD4 lymphocytes, while certain chemokines act selectively on naive T cells (23) or memory CD4 lymphocytes (24). Furthermore, several chemokines that interact with these receptors have been shown to be expressed in inflammatory sites (25) and lymph nodes (26). Within the memory CD4 lymphocyte population are subsets of effector T lymphocytes, Th1 and Th2, classified by the cytokines they secrete (27). Th1 lymphocytes secrete cytokines such as lymphotoxin and IFN-{gamma} to promote cellular immune responses, while Th2 lymphocytes release cytokines such as IL-4 and IL-5 to promote the humoral immunity and allergic responses (28). Recently, it has been shown that in vitro derived Th1/Th2 lymphocyte lines differentially express chemokine receptors. CXCR3 and CCR5 are selectively expressed by in vitro derived Th1 lymphocytes, while CCR4, CCR8, and CCR3 are expressed on Th2 lymphocytes (24, 29, 30, 31, 32, 33). Interestingly, the chemokines macrophage-derived chemokine (MDC) (stimulated T cell chemoattractant protein-1) for CCR4 and IFN-{gamma}-inducible protein-10 for CXCR3 are induced by cytokines associated with Th1 and Th2 environments, respectively (24, 34, 35). Furthermore, Imai et al. (33) showed that T cells attracted by thymus and activation-regulated chemokine (TARC) and MDC predominantly produce Th2-type cytokines, and that fractionated CCR4+ cells also preferentially produce Th2-type cytokines.

In the studies reported in this work, by using specific anti-CCR4 mAbs, we confirm that CCR4 is differentially expressed on in vitro derived Th1/Th2 effector lymphocytes and demonstrate that CCR4 is the major chemokine receptor on CD4 lymphocytes through which MDC and TARC act. Moreover, we define a large subset of memory CD4 lymphocytes that express CCR4, which comprise ~50% of memory CD4+ cells, including nearly all CLA+ cells. A subset of CCR4+ cells coexpresses CXCR3 and CCR5 that are associated with in vitro derived Th1 cells. Although Th2 cytokine production is linked with expression of CCR4, CCR4+ memory T cells also contain non-Th2 cells expressing IFN-{gamma}. Therefore, contrary to the preferential expression pattern of CXCR3, CCR5, and CCR4 on T cells that are generated in vitro in polarized conditions, CCR4 cannot specifically mark Th2 populations in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of CCR4 stable transfectants and other transfectants

CCR4 cDNA was obtained by RT-PCR on RNA isolated from a basophilic cell line KG1a using a 5'-oligonucleotide primer and a 3'-oligonucleotide primer, which correspond to the CCR4 sequences and contain flanking HindIII and XbaI sites, respectively. The PCR fragment was subcloned into the HindIII and XbaI sites of pMRB101, in which the inserted gene expression was driven by a CMV promoter. The DNA was stably transfected into a murine pre-B lymphoma cell line (L1.2), as described (36, 37, 38). The cells expressing high levels of CCR4 were selected by serial dilution/subcloning for their ability to chemotax to TARC and MDC. To enhance the receptor expression, the cells were treated at 106/ml with 5 mM butyric acid for 16–18 h before the experiment.

Other cell lines used included stable transfectants of the L1.2 murine pre-B lymphoma cells expressing CCR1–3, CCR5–9, CXCR1-CXCR5, as well as transfectants expressing various orphan G protein-coupled receptors, including BONZO, BOB, LyGPR, APJ, RDC, GPR5, GPR9-6, and AF014958.

Purification of cell populations

Venous blood was collected from volunteer donors, and PBMC were isolated by Ficoll-Hypaque density-gradient centrifugation, as described (36). To isolate different subsets of leukocytes, human peripheral blood was collected in 10% (v/v) 0.1 M EDTA, layered onto 1-Step Polymorphs gradient (Accurate Chemicals, Westbury, NY), and centrifuged at 400 x g for 30 min at room temperature. Neutrophil and mononuclear cell layers were collected, resuspended in Dulbecco’s PBS without calcium and magnesium (Life Technologies, Grand Island, NY), and centrifuged for 15 min at ~750 x g. RBC were lysed in the neutrophil fraction by resuspending the pellet in E-Lyse (Cardinal Associates, Santa Fe, NM) for 5 min on ice. Both cell fractions were washed twice with ice-cold Dulbecco’s PBS. The mononuclear cells were allowed to adhere to protein-coated plastic for 2–3 h, and then nonadherent cells were gently washed off the plate. CD4 cells were purified from mononuclear cells with the relevant Miltenyi beads using 20 µl of beads for 107 mononuclear cells in PBS/1% BSA/5 mM EDTA at 5 x 107 cells/ml for 30 min at 4°C. They were then spun down, resuspended in PBS/1% BSA/5 mM EDTA at 5 x 107 cells/ml, and passed over a VS column (Miltenyi Biotech, Auburn, CA) in a magnetic field to remove nontagged cells. Cells were removed by forcing 20 ml of PBS/1% BSA/5 mM EDTA over the VS column, outside the magnetic field.

Preparation of chronically activated Th1 and Th2 lymphocytes

As previously described (39), six-well Falcon plates were coated overnight with 10 µg/ml anti-CD28 and 2 µg/ml OKT3, and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, Germantown, MD) were cultured at 105-106 cells/ml in DMEM with 10% FCS and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL-4 (1 µg/ml) were used to direct to Th1, while IL-4 (5 ng/ml) and anti-IFN-{gamma} (1 µg/ml) were used to direct to Th2. After 4–5 days, the activated Th1 and Th2 lymphocytes were washed once in DMEM and expanded for 4–7 days in DMEM with 10% FCS and IL-2 (1 ng/ml). Following this, the activated Th1 and Th2 lymphocytes were restimulated for 5 days with anti-CD28/OKT3 and cytokines, as described above, but with the addition of anti-CD95L (1 µg/ml) to prevent apoptosis. After 4–5 days, the Th1 and Th2 lymphocytes were washed and then expanded again with IL-2 for 4–7 days. Activated Th1 and Th2 lymphocytes were maintained in this way for a maximum of three cycles.

Generation of anti-CCR4 mAbs

C57/Black mice were immunized with 107 CCR4/L1.2 transfectants per mouse at 2- to 3-wk intervals for 6–12 times. For the first three immunizations, the transfectants were pretreated with mitomycin C for 30 min before washing and injection to prevent tumor growth in the mice. Spleens were harvested from mice 3–4 days after the last immunization, and cells were fused with the SP2/0 cell line, as described (40). Generally, 3000–5000 hybridomas were generated per fusion and the supernatants were tested for differential staining of CCR4/L1.2 transfectants and other chemokine receptor L1.2 transfectants. Of the 15 fusions performed, two fusions gave rise to anti-CCR4 mAbs, 1G1 (isotype IgG1) and 2B10 (isotype IgG2a), respectively.

Other Abs and reagents

Anti-CD4, -CD8, -19, -49d, -56, -62L, -CLA, -CD45RA, and -CD45RO dye-linked mAbs for immunofluorescence studies were all obtained from PharMingen (San Diego, CA). OKT3, an anti-human CD3 mAb, was obtained from American Type Culture Collection (ATCC, Manassas, VA), and anti-human CD28 mAb was purchased from Becton Dickinson (San Diego, CA). The majority of the anti-chemokine receptor mAbs were generated in LeukoSite (Cambridge, MA) and have the clone names 1D9 (CCR2), 7B11 (CCR3), 2D7 (CCR5), 11A9 (CCR6), and 1C6 (CXCR3). Recombinant human chemokines were obtained from PeproTech (Rocky Hill, NJ) and R&D Systems (Minneapolis, MN). The human endothelial cell line ECV304 was purchased from ATCC. All cytokines were obtained from R&D Systems.

125I-labeled human TARC (125I-TARC) and MDC (125I-MDC) binding

125I-TARC and 125I-MDC were purchased from DuPont-NEN (Boston, MA) and Amersham (Arlington Heights, IL), respectively, and unlabeled chemokines were obtained from PeproTech or R&D Systems. Chemokine binding to target cells was conducted using the following procedure: CCR4/L1.2 cells were washed and resuspended in binding buffer (50 mM HEPES, pH 7.5, 1 mM CaCl2, 5 mM MgCl2, and 0.5% BSA) at 107/ml. For each binding reaction (in a final volume of 100 µl), 25 µl of cell suspension (2.5 x 105 cells) was mixed with 0.1 nM radiolabeled chemokine with or without an appropriate amount of anti-CCR4 mAb or an isotype-matched control mAb. Total binding was in the presence of radiolabeled chemokines only, and nonspecific binding (background) was determined in the presence of 100 nM unlabeled chemokines. The reactions were incubated at room temperature for 45–60 min, and stopped by transferring the mixture to Unifilter-96 GF/B (Packard) plates that were then washed two to three times with binding buffer containing 0.5 M NaCl. The plates were dried and MicroScint scintillation fluid was added before counting. Each sample was assayed in duplicate.

Chemotaxis assay

The 3-µm-pore-diameter Transwell inserts were used either uncoated or coated with 2% gelatin for 2 h. Then 0.45 ml of DMEM with 5% FCS was placed in the lower wells of the chambers, and 2 x 105 ECV304 cells were added to each gelatin-coated insert in 0.2 ml of DMEM/5% FCS. After 2 days, the wells and inserts were washed twice with RPMI containing 0.5% BSA and 10 mM HEPES, and then chemokine was added to the lower well. For chemotaxis of transfectant cells, no ECV304 cells were used to coat the plates. The cells under study were washed once in RPMI and resuspended at 4 x 106 cells/ml for Th1/Th2 lymphocytes and cell lines and at 107 cells/ml for transfectants and resting CD4 lymphocytes in RPMI containing 0.5% BSA and 10 mM HEPES. An aliquot of 200 µl of cell suspension (input of 8 x 105 cells and 2 x 106 cells, respectively) was added to each insert. After 2–4 h, the inserts were removed and the number of cells that had migrated through the ECV304 monolayer to the lower well were counted for 30 s on a Becton Dickinson FACScan with the gates set to acquire the cells of interest. Using this technique, 100% migration would be 25,000 cells for Th1/Th2 cells and 75,000 cells for resting CD4 lymphocytes, in which this number represents the cells in the lower well counted on the FACScan for 1 min. To study the phenotype of migrating cells, identical experiments with CD4 lymphocytes were performed with six-well plates using 24-mm-diameter inserts. Chemotaxis assays were identical with ECV304 migration assays, but fibronectin-coated inserts (10 µg/ml) were used. In all cases, the data points were the result of duplicate wells, with the mean value shown and the error bars representing the sample SD.

Intracellular cytokine staining

Mononuclear cells from human peripheral blood were prepared by centrifugation on Histopaque-1077 (Sigma, St. Louis, MO). Untouched CD4+ T cells (purity >97%) were isolated by depleting non-CD4+ T cells using a magnetic bead depletion method (Miltenyi Biotec). CD4+ T cells were stained with Abs to CLA, CD45RA, and CCR4. CD45RA+ (naive) CCR4- CLA-, CD45RA- (memory) CCR4- CLA-, CD45RA- CCR4+ CLA-, and CD45RA- CCR4+ CLA+ cells were sorted using FACStar (Becton Dickinson, San Jose, CA). Sorted cells were activated for 5 h at 37°C with PMA (50 ng/ml) and ionomycin (1 µg/ml) in RPMI 1640 medium supplemented with penicillin/streptomycin, 10% FBS, 50 U/ml IL-2, and 1 µg/ml brefeldin A. Activated cells were fixed and permeabilized using Cytofix/Cytoperm solution (PharMingen) and stained with PE-conjugated isotype control Abs or mAbs to IL-2, IL-5, IFN-{gamma} (from PharMingen), and IL-4 (Becton Dickinson). Data were analyzed on FACSCaliber using CellQuest program (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mAbs 1G1 and 2B10 specifically react with CCR4/L1.2 transfectants

Two mAbs were raised in C57/Black mice to CCR4/L1.2 transfectants. Both mAbs reacted with the CCR4/L1.2 transfectants, but not with parental L1.2 cells. The specificity of these two mAbs for CCR4 was further extensively examined using a large panel of L1.2 transfectants. Neither 1G1 nor 2B10 cross-reacted with L1.2 transfectants expressing CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, and CCR8 (shown in Fig. 1Go for 1G1). They also did not cross-react with L1.2 transfectants expressing CXCR1-CXCR5, or orphan G protein-coupled receptors including BONZO, BOB, LyGPR, APJ, RDC, GPR5, GPR9-6, and AF014958 (not shown).



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  		FIGURE 1. Identification of CCR4-specific mAbs. Stable L1.2 transfectants expressing either CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, or CCR8 were stained with mAb 1G1. Negative control staining for all the L1.2 transfectants resembled the staining shown for 1G1 on CCR1/L1.2 cells. mAb 2B10 showed the same pattern of expression.

 
CCR4 is expressed on subsets of CD4 lymphocytes and a minor subset of CD8 lymphocytes in peripheral blood

Both 1G1 and 2B10 were used to examine the CCR4 expression on leukocyte subpopulations, and similar results were obtained with both mAbs. Anti-CCR4 mAb 1G1 was used in the FACS experiments described in this work. In initial two-color studies of peripheral blood, CCR4 was found to be expressed on a large subset (20–30%) of CD4 lymphocytes as well as on a smaller subset (3–5%) of CD8 lymphocytes (Fig. 2GoA). Although CCR4dull and CCR4high subsets of CD4 lymphocytes were evident, CCR4 was expressed at low levels on CD8 lymphocytes. B lymphocytes, basophils, eosinophils, neutrophils, and NK cells did not express CCR4 (Fig. 2GoA and data not shown). As would be predicted from its expression on blood T lymphocytes, in examining a large panel of cell lines, CCR4 was found only on T cell lines (Peer and CEM cells, data not shown).



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  		FIGURE 2. CCR4 expression on peripheral blood leukocytes. A, Isotype-matched mAb control staining. B, Expression of CCR4 on freshly isolated PBMCs. A two-color staining protocol was used to assess expression of CCR4 and the cell markers, CD4 and CD8 (T cells), CD19 (B cells), and CD56 (NK cells). C, CCR4 expression on CD4+ T cells. A three-color staining protocol was used to assess the expression of CCR4 and the T cell subset markers, CD45RA (naive), CLA (skin homing), and {alpha}4{beta}7 (gut homing), CD62L (peripheral lymph node homing), and CD49d on CD4+ T cells. The cells were analyzed by gating on CD4+ lymphocytes. CCR4 expression was shown in x-axis in all plots, and the cell marker expression was shown in y-axis and indicated in each plot. Quadrants were set according to the staining of control mAbs, and the staining was representative of multiple donors analyzed. All results shown were using mAb 1G1 (n = 3), and similar results were obtained when mAb 2B10 was used.

 
We then examined the subset of CD4 lymphocytes that express CCR4 in greater detail by three-color staining (Fig. 2GoB). The CD4 lymphocytes that expressed CCR4 were mostly of memory phenotype CD45RA-. As reported in our recent studies (41), memory CLA+ CD4 lymphocytes, which traffic to skin, were all CCR4high. However, CCR4high CLA- and CCR4dull CLA- CD4 lymphocytes were also evident. In contrast, the subset of memory {alpha}4{beta}7high CD4 lymphocytes, which traffic to mucosal sites, expressed little or no CCR4. Moreover, the subset of memory CD4 lymphocytes that expressed the highest level of CCR4 was predominantly {alpha}4{beta}7-. Furthermore, the subset of memory CD4 lymphocytes that lacks {alpha}4 integrins ({alpha}4{beta}1- {alpha}4{beta}7-) all expressed high levels of CCR4 (Fig. 2GoB). CD62L was expressed on both CCR4+ and CCR4- subsets of memory CD4 lymphocytes.

To further correlate the expression of CCR4 on CD4 lymphocytes with its function, we examined the subset of CD4 lymphocytes that chemotax to MDC by FACS. We used the anti-CCR4 mAb 1G1 in this experiment, as binding of 2B10 to CD4 lymphocytes was inhibited by MDC (data not shown). CCR4+ CD4 lymphocytes were enriched in the population of CD4 lymphocytes that chemotax to MDC (2.6-fold). We also saw an enrichment for CLA+ (4.5-fold) and CD45RA- memory CD4 lymphocytes (2.9-fold) in the migrated population. Among CCR4+ memory CD4 lymphocytes, the majority of the migration to MDC was in the CLA+ CCR4+ subset rather than the CLA- CCR4+ subset (4.5 vs 1.6). Finally, in examining the chemotaxis of 16 cell lines to MDC and TARC, we found that only the two cell lines that expressed CCR4 chemotaxed to MDC and TARC (data not shown). Therefore, expression of CCR4 on T lymphocytes and functional response to MDC/TARC correlate.

CCR4 is expressed on a subset of mature single-positive (SP) CD4+ thymocytes

Because CCR4 transcripts are present in thymus, we examined the expression of CCR4 on thymocytes. CCR4 was expressed on a small subset (14%) of thymocytes (Fig. 3GoA). The expression of CD3 on all CCR4+ thymocytes indicates that CCR4 is up-regulated during thymocyte maturation. Another chemokine receptor, CXCR4, was expressed on immature thymocytes, while CCR9 was expressed at all stages of T cell development. Due to the expression of CCR4 on mature thymocytes, we analyzed expression of this chemokine receptor in immature double-negative CD4-CD8- and double-positive CD4+CD8+ thymocytes as well as mature SP CD4 and CD8 thymocytes (Fig. 3GoB). As expected, CCR4 expression was restricted to mature SP thymocytes and was mainly expressed on SP CD4 thymocytes. The majority of SP CD4 thymocytes express CCR4, but a small subset of CCR4- CD4 SP thymocytes was also evident.



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  		FIGURE 3. CCR4 is expressed on a subset of thymocytes that express high levels of CD3 and are predominantly SP CD4+. Thymocytes isolated from children undergoing surgery were stained in A, two-color experiments using anti-CCR4 mAb 1G1, followed by F(ab')2 anti-mouse IgG PE to study expression of CCR4 on thymocytes expressing varying levels of CD3, using anti-CD3 tricolor to determine expression of CD3. Staining with an IgG1 control mAb was used to establish background staining, while anti-CXCR4 and anti-GPR-9-6 were used as positive control mAbs. B, Thymocytes were stained in three-color experiments with anti-CCR4 mAb 1G1, followed by F(ab')2 anti-mouse IgG PE, using anti-CD8 PE and anti-CD4 tricolor to define the four stages of T cell development: CD4-CD8-, CD4+CD8+, CD4+, and CD8+. Experiments were performed four times with similar results.

 
mAbs 1G1 and 2B10 block the binding of MDC and TARC to CCR4

The ability of the mAbs 1G1 and 2B10 to inhibit the binding of TARC (Fig. 4GoA) and MDC (Fig. 4GoB) to CCR4 was examined. Both mAbs blocked the 125I-TARC and 125I-MDC binding to CCR4/L1.2 transfectants, while the isotype-matched control mAbs had no significant effect. Furthermore, both 1G1 and 2B10 effectively blocked the TARC- and MDC-mediated chemotaxis in CCR4/L1.2 transfectants (Fig. 4Go, C and D). Of greater interest, 2B10 and to some extent 1G1 significantly inhibited chemotaxis of resting CD4 lymphocyte (Fig. 5GoA) and Th2 lymphocyte (Fig. 5GoB) to TARC and MDC. Both mAbs had no effect on the chemotaxis of CD4 lymphocytes to RANTES.



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  		FIGURE 4. mAbs 1G1 and 2B10 inhibit the TARC- and MDC-mediated functions in CCR4/L1.2 cells. A and B, Inhibition of 1G1 and 2B10 in binding of 125I-TARC and 125I-MDC to CCR4/L1.2 cells. CCR4/L1.2 cells (2 x 105) were pretreated with 20 µg/ml mAbs 1G1, 2B10, isotype-matched control mAbs, or 100 nM unlabeled TARC or MDC for 10 min on ice before addition of 125I-TARC (A) or 125I-MDC (B). After 1-h incubation at room temperature, the cells were washed and counted, as described in Materials and Methods. C and D, Inhibition of CCR4/L.12 chemotaxis to TARC and MDC by mAb 1G1 and 2B10. L1.2/CCR4 transfectants (4 x 105 cells) preincubated with various amounts of 1G1 or 2B10 were placed in the top chamber of the transwell plate, and 12 nM TARC (C) or MDC (D) was placed in the bottom. After 3–5 h, the number of cells migrating to the bottom wells was counted and divided by the number of cells that migrated to 12 nM MDC or TARC when pretreated with an IgG1 control to determine the percentage of maximum migration. The chemotaxis in the presence of an IgG1 control was not significantly different from chemotaxis to chemokine alone. The experiment was performed three times with similar results.

 


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  		FIGURE 5. CCR4 is a major receptor for TARC- and MDC-mediated responses in Th2 lymphocytes and resting memory T cells. A, Inhibition of Th2 lymphocyte chemotaxis to TARC and MDC by anti-CCR4 mAbs 1G1 and 2B10. B, Inhibition of T lymphocyte chemotaxis to MDC and TARC by 1G1 and 2B10. For these experiments, cells were allowed to chemotax to TARC, MDC, or RANTES in the presence or absence of mAb 1G1, mAb 2B10, or an irrelevant control mAb in a transwell chemotaxis assay. Cells (4 x 106) preincubated with 50 µg/ml mAb 1G1, mAb 2B10, or control mAb were placed in the top chamber of the transwell plate, and chemokine was placed in the bottom. After 2 h, the number of cells migrating to the bottom was counted using flow cytometry. Similar results were obtained in two experiments.

 
Activation of naive T cells in Th2 polarization condition is required for CCR4 expression on most T cells

Although umbilical CD4 lymphocytes did not express CCR4 (data not shown), chronic activation of these cells in the presence of IL-4 to generate Th2 lymphocytes resulted in CCR4 expression (Fig. 6Go), while chronic activation with IL-12 to generate Th1 lymphocytes did not up-regulate CCR4 expression. As expected, CXCR3 was up-regulated on Th1 lymphocytes, while {alpha}4{beta}7, an integrin used in lymphocyte trafficking to mucosal sites, and chemokine receptor CCR2 were up-regulated on both Th1 and Th2 lymphocytes (Fig. 6Go).



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  		FIGURE 6. CCR4 is expressed selectively on in vitro derived Th2 cells. Chronically activated Th1 and Th2 effector lines were established by stimulation of umbilical CD4 lymphocytes with plate-bound anti-CD3 and anti-CD28, as described in Materials and Methods. After two rounds of stimulation, the Th1 (line profile) and Th2 (filled profile) effector cell lines were expanded in IL-2 for 4 days and then stained with anti-CCR2 mAb 1D9, anti-{alpha}4{beta}7 mAb ACT-1, anti-CXCR3 mAb (R and D), and anti-CCR4 mAb 1G1, followed by F(ab')2 anti-mouse IgG PE. The experiment was performed six times with similar results.

 
Activation of T lymphocytes with anti-CD3 mAb resulted in transient down-regulation of CCR4 over 2–3 days, with expression recovering upon cessation of TCR cross-linking and culture in the presence of IL-2 (data not shown). This is similar to what was observed for CCR5, CCR6, and CXCR3. The CCR4 expression peaks between 4 and 8 days after activation, and declines thereafter.

CCR4 is coexpressed with Th1-associated chemokine receptors CCR5 and CXCR3

The expression of CCR4 on CD4 lymphocytes in relation to other chemokine receptors known to be expressed on subsets of memory CD4 lymphocytes was examined in three-color experiments (Fig. 7Go). CCR4 was found on both positive and negative subsets of CCR2 and CCR6 CD4 lymphocytes. Surprisingly, given the association of CCR5 and CXCR3 expression with Th1 phenotype by in vitro studies, a significant population of CCR5+CCR4+ and CXCR3+ CCR4+ CD4 lymphocytes was observed in these experiments.



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  		FIGURE 7. CCR4 is coexpressed with Th1-associated chemokine receptors CCR5 and CXCR3. Mononuclear cells were stained in a three-color study with 1G1, followed by F(ab')2 anti-mouse IgG FITC. CD4 lymphocytes were marked with anti-CD4 tricolor, while subsets of cells and anti-CCR6, anti-CCR5, anti-CCR2, and anti-CXCR3 mAbs linked to PE were used to study the coexpression of these chemokine receptors with CCR4. The profiles shown represent the coexpression of CCR2,5,6 and CXCR3 on CD4 lymphocytes, and gated on CD4+ cells. The experiment was performed twice with similar results.

 
Production of Th1 and Th2 cytokines by CCR4+ CLA+, CCR4+ CLA-, and CCR4- memory CD4 lymphocytes

As CCR4 is coexpressed on memory CD4 lymphocytes with Th1-associated chemokine receptors, we next examined the cytokines produced by activated subsets of memory CD4 lymphocytes defined by expression of CCR4 and CLA. As expected, naive and memory CD4 lymphocytes secreted IL-2 on stimulation, while production of IL-4, IL-5, and IFN-{gamma} was restricted to the memory CD4 lymphocytes (Fig. 8GoA). Production of IFN-{gamma} was associated with the CCR4- memory CD4 lymphocytes, with 51% of CCR4- CLA- CD4 memory cells producing IFN-{gamma} on activation, while only 17% of the CCR4+ CLA- and 10% of the CCR4+ CLA+ memory CD4 lymphocytes produced this cytokine. Similarly, production of Th2 cytokines IL-4 and IL-5 was positively associated with CCR4 expression on CD4 lymphocytes, with the CLA- CCR4+ memory CD4 lymphocytes being the major producers of these two cytokines. However, while Th2 and Th1 cytokine production was positively and negatively associated with CCR4 expression, respectively, expression of IFN-{gamma} was observed in significant (5–30%) subsets of activated CCR4+ memory CD4 lymphocytes, and IL-4 production was also observed in a subset (up to 3%) of freshly isolated CCR4- memory CD4 lymphocytes on activation. Studies of additional normal donors for IL-2, IL-4, and IFN-{gamma} production were also performed and similar results were observed (Fig. 8GoB).



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  		FIGURE 8. Expression of effector cytokines by memory T cell subsets defined by expression of CCR4 and CLA expression. Peripheral blood CD4+ T cell subsets were isolated, activated for 5 h with PMA and ionomycin, and analyzed for cytokine expression, as described in Materials and Methods. One representative set of data from three independent experiments is shown (A). Data from six different donors are plotted to show trends and individual variability of IL-2, IL-4, and IFN-{gamma} expression in memory T cell subsets (B). *, Indicates significant difference from CCR4- CD4 T cells (p < 0.02, Student’s t test).

 
To increase the percentage of cells producing Th2 cytokines, sorted CCR4+ and CCR4- memory CD4 lymphocytes were activated with anti-CD3 and expanded in IL-2 before performing activation and intracellular staining. Larger percentages of cells produced IL-4, IL-5, and IL-13, and similar results were obtained for CCR4-/CCR4+ CD4 lymphocyte subset production of these cytokines: IL-4 (2.4%/12.5%), IL-5 (2.8%/14%), IL-13 (2%/24%), and IFN-{gamma} (81%/24%).

Finally, we sorted CCR4+/-, CLA+/-, CD45RA+/- CD4 T cell subsets and stimulated them with anti-CD28 and anti-CD3 in nonpolarizing culture condition (without added exogenous IL-4 or IL-12 or other neutralizing Abs) for 4 days and then cultured them in IL-2 for 3 days (the first activation cycle). Half the cultured cells were examined for intracellular cytokine analysis, and the other half were cultured again (the second activation cycle) and examined for their cytokine production. Although in vitro differentiated CCR4+ and CCR4- CD4 T cells maintained their relative differences in production of IL-4 or IFN-{gamma}, CCR4+ CD4 T cells after two cycles of activation contained not only more T cells producing IL-4, but also included more T cells producing IFN-{gamma} than fresh CD4 T cells (Fig. 9Go). This is also the case for the CCR4- CD4 T cells. These combined data suggest that CCR4+ CD4 T cells have potentials to become Th1 as well as to become Th2 cells.



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  		FIGURE 9. CCR4+ CD4 T cells differentiate into cells producing IFN-{gamma} as well as cells producing IL-4 after activation with anti-CD3 and CD28 Abs. Sorted memory (CD45RA-) CCR4+ CLA+, CCR4+ CLA-, and CCR4- CD4 T cell subsets were activated with anti-CD28 and anti-CD3 in IL-2 (100 U/ml, without added exogenous IL-4 or IL-12 or other neutralizing Abs) for 4 days and then cultured in IL-2 (100 U/ml) for 3 days (this is the first activation cycle). Half the cultured cells were examined for intracellular cytokine analysis, and the other half were cultured again (the second activation cycle) and examined for their cytokine production. Data shown are one representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown in this study that CCR4 is expressed on a subset of memory CD4+ lymphocytes, a very small subset of CD8+ lymphocytes, and thymocytes. Moreover, we have demonstrated that CCR4 is the major memory T lymphocyte trafficking receptor for MDC and TARC. Finally, we provide evidence to suggest that despite its preferential expression on in vitro derived Th2 cells, CCR4+ memory T cell subset also contains non-Th2 populations in vivo.

Recently, TARC was reported to bind to CCR8 (42). However, in our hands and in other reports (43), TARC and MDC mediated only chemotaxis of CCR4 transfectants and not other chemokine receptor transfectants, including CCR8. A screening of a panel of cell lines in this study indicates that only CCR4+ cell lines chemotax to MDC and TARC. In addition, the majority of chemotaxis of both resting CD4+ cells and chronically activated Th2 lymphocytes to MDC and TARC can be blocked by anti-CCR4 mAbs. We also found that the subset of CD4 lymphocytes that migrated to MDC is enriched for CCR4-expressing cells. These combined results suggest that MDC and TARC predominantly use CCR4 on T cells in mediating chemotaxis of CD4 lymphocytes.

Our studies demonstrate that CCR4 is expressed on a large subset of the SP CD4+ thymocytes that express high levels of TCR, suggesting CCR4 expression is induced on thymocytes at a late stage of T cell development. The restricted expression of CCR4 on SP CD4+ thymocytes correlates with the chemotaxis of this subset of thymocytes to MDC and further demonstrates that expression of CCR4 and chemotaxis to MDC correlate (44). TARC and MDC are also reported to be expressed in the thymus (45, 46) and by APCs (24, 47). It is possible that TARC and MDC are expressed on macrophages, dendritic cells, and epithelial cells in the thymus and attract mature CCR4+ CD4 SP thymocytes to modulate positive and negative selection during T cell development. Interestingly, CD45RA naive T lymphocytes in the periphery do not express CCR4. Therefore, on exit of SP CD4 thymocytes from the thymus to the periphery, CCR4 expression must be down-regulated. Alternatively, those SP CD4 thymocytes that do not express CCR4 in the thymus may represent fully mature thymocytes about to exit the thymus.

The subset of memory CD4 lymphocytes that express CCR4 encompasses nearly all CD4 lymphocytes that express CLA, but not {alpha}4{beta}7. Indeed, nearly all CLA+ {alpha}4{beta}7- memory CD4 lymphocytes express high levels of CCR4. However, most importantly, CCR4 is expressed at high levels by a subset of CLA- {alpha}4{beta}7- cells, suggesting that CCR4 may mediate other functions in addition to its proposed role in trafficking of CLA+ memory CD4 lymphocytes to skin. It was shown previously that CLA was expressed by T cells responsible for the Th2 cytokine production in individuals with skin allergy (48, 49). Nevertheless, in individuals with allergy at nonskin sites, Th2 lymphocytes were also found in the memory CLA- CD4 subset. The expression of MDC by APCs, including activated B cells and the induction of MDC as well as CCR4 on T lymphocytes by Th2 cytokines (21, 32), indicates that CCR4 is possibly also involved in local movement of memory CD4 lymphocytes in lymphoid tissue toward dendritic cells and B cells. Finally, the expression of CCR4 on both CD62L+ and CD62L- memory CD4 lymphocytes indicates that the memory CCR4+ CD4 subset includes both effector CD62L- memory CD4 lymphocytes that are thought to traffick principally to inflammatory sites as well as resting memory CD62L+ CD4 lymphocytes that can most likely also traffick to secondary lymphoid organs such as peripheral lymph nodes.

It was previously shown that MDC and TARC specifically act on in vitro derived effector Th2 lymphocytes, while CCR4 transcripts are differentially expressed by in vitro derived Th1/Th2 lymphocytes (24, 30). Consistent with these observations, we and Imai et al. (33) found that CCR4 protein is preferentially expressed on the surface of in vitro derived Th2 cells. However, if CCR4 were a specific marker for Th2 cells, one would have predicted that CCR4 would be expressed by a very small subset of memory CD4 lymphocytes, as effector Th2 cells represent only a small subset of peripheral blood CD4+ cells. Therefore, the demonstration in this study that CCR4 is expressed on a large subset of circulating memory CD4 lymphocytes was surprising. As MDC blocks all staining of leukocytes by the anti-CCR4 mAb 2B10 and both our anti-CCR4 mAbs stain identical subsets (data no shown), we are confident that our results represent the distribution of CCR4 on leukocytes. Imai et al. also showed recently that, using an independent anti-CCR4 mAb, CCR4 was detected on ~20% CD4+ T cells (33), although the intensity of their staining was weaker than what we obtained using both of our mAbs.

Our experiments examining coexpression of CCR4 with other chemokine receptors on CD4 lymphocytes also gave unexpected results. We found that CCR4 was expressed on a significant subset of CD4 lymphocytes that also express chemokine receptor CCR5 and CXCR3, both of which are selectively expressed on in vitro derived effector Th1 lymphocytes. More importantly, our data suggest that although CCR4 behaves like a specific Th2 marker on in vitro derived Th2 lymphocytes, it does not specifically mark the Th2 population in circulating blood, and in this sense, neither do CCR5 and CXCR3 specifically mark the Th1 population. CCR4+ CD4 lymphocytes may represent a larger subset of memory CD4 lymphocytes, within which are contained the majority of Th2 lymphocytes. Alternatively, the culture conditions used to generate in vitro derived Th2 lymphocytes may induce expression of CCR4. In further studies, we directly examined the cytokines produced by memory CD4 lymphocytes from blood sorted for expression of CCR4 and CLA. Although IL-4-producing cells were more frequent in the CCR4+ than CCR4- subsets, there tended to be a greater frequency of IFN-{gamma}-producing Th1 cells among the CCR4- subset. Furthermore, only a small subset of memory CCR4+ CD4 lymphocytes produces Th2 cytokines on activation. When freshly sorted CCR4+ and CCR4- CD4 lymphocytes were activated and expanded before performing intracellular cytokine staining, the Th2 correlation was still observed. It is possible that in certain chronic inflammation conditions, the linkage of CCR4 and Th2 phenotype may increase depending on cytokine expression profiles of tissue microenvironment. Further studies in patients with Th2 inflammatory diseases, such as atopic dermatitis and asthma, are underway to investigate this.

Differences in the results obtained from in vitro derived Th1/Th2 lymphocytes and freshly isolated T lymphocytes in blood have also been observed for expression of adhesion molecules, such as CLA. Although CLA is selectively expressed on in vitro derived Th1 lymphocytes (50), in vivo studies in both normal and atopic dermatitis patients have yielded conflicting data. Under normal conditions, the CLA subsets of memory CD4 lymphocytes produce both Th1 and Th2 cytokines (51), while in atopic dermatitis the CLA subset contains predominantly Th2 lymphocytes (48). In conclusion, our data would indicate that in normal patients there is a linkage of CCR4 with Th2 phenotype, but that this is not at all a tight linkage. Most importantly, our results indicate that this linkage is malleable, and suggest that effector cytokine production and patterns of chemokine receptor expression (at least of CCR4, CXCR3, and CCR5) can be independently controlled and regulated in vivo, thus optimizing the potential for phenotypic control of subset-specific homing to particular sites such as skin.

In summary, in this study we show that CCR4 is expressed on a small subset of mature SP CD4+ thymocytes and a major subset of CD4 lymphocytes, which contains all CLA+ {alpha}4{beta}7- CD4 lymphocytes, as well as a small population of CLA- memory CD4 lymphocytes. Chemotaxis of CD4 lymphocytes to MDC and TARC correlates with expression of CCR4 and is blocked by anti-CCR4 mAbs. Therefore, CCR4 represents the major chemokine receptor through which TARC and MDC act on memory CD4 lymphocytes. Our data indicate that in peripheral blood, subsets of memory CD4 lymphocytes are present that express CCR4 as well as CXCR3 or CCR5. Also, although there is a linkage between Th2 phenotype and CCR4 expression in normal blood, distinct populations of CCR4+ memory CD4 lymphocytes that produce IFN-{gamma} on activation are evident. Therefore, our data suggest that the refinement of Th1 and Th2 is more complex in vivo than in vitro. Chemokine receptors may play distinct roles at different stages of memory effector T cell differentiation, very likely reflecting the different influence and refinement of local immune responses as a function of tissues, microenvironment, and cytokine milieu.


   Acknowledgments
 
We are grateful to Walter Newman for reading the manuscript and Nasim Kassam and Zhimin Shen for technical assistance.


   Footnotes
 
1 D.P.A., N.R., and C.H.K. contributed equally to this work. Back

2 C.H.K is a Leukemia Society of America fellow. Back

3 Address correspondence and reprint requests to Dr. Lijun Wu, Millennium Inc., 75 Sidney Street, Cambridge, MA 02139. Back

4 Abbreviations used in this paper: CLA, cutaneous lymphocyte Ag; CXCR, CXC chemokine receptor; 125I-MDC, 125I-labeled MDC; 125I-TARC, 125I-labeled TARC; MDC, macrophage-derived chemokine; SP, single positive; TARC, thymus and activation-regulated chemokine. Back

Received for publication March 17, 2000. Accepted for publication September 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Taub, D. D., J. J. Oppenheim. 1994. Chemokines, inflammation and the immune system. Ther. Immunol. 1:229.[Medline]
  2. Locati, M., P. M. Murphy. 1999. Chemokines and chemokine receptors: biology and clinical relevance in inflammation and AIDS. Annu. Rev. Med. 50:425.[Medline]
  3. Baggiolini, M., B. Dewald, B. Moser. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15:675.[Medline]
  4. Baggiolini, M.. 1998. Chemokines and leukocyte traffic. Nature 392:565.[Medline]
  5. Taub, D. D., M. Anver, J. J. Oppenheim, D. L. Longo, W. J. Murphy. 1996. T lymphocyte recruitment by interleukin-8 (IL-8): IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J. Clin. Invest. 97:1931.[Medline]
  6. Fuentes, M. E., S. K. Durham, M. R. Swerdel, A. C. Lewin, D. S. Barton, J. R. Megill, R. Bravo, S. A. Lira. 1995. Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1. J. Immunol. 155:5769.[Abstract]
  7. Bargatze, R. F., E. C. Butcher. 1993. Rapid G protein-regulated activation event involved in lymphocyte binding to high endothelial venules. J. Exp. Med. 178:367.[Abstract/Free Full Text]
  8. Sekido, N., N. Mukaida, A. Harada, I. Nakanishi, Y. Watanabe, K. Matsushima. 1993. Prevention of lung reperfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature 365:654.[Medline]
  9. Karpus, W. J., N. W. Lukacs, B. L. McRae, R. M. Strieter, S. L. Kunkel, S. D. Miller. 1995. An important role for the chemokine macrophage inflammatory protein-1{alpha} in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
  10. Gupta, S. K., T. Hassel, J. P. Singh. 1995. A potent inhibitor of endothelial cell proliferation is generated by proteolytic cleavage of the chemokine platelet factor 4. Proc. Natl. Acad. Sci. USA 92:7799.[Abstract/Free Full Text]
  11. Broxmeyer, H. E., C. H. Kim. 1999. Regulation of hematopoiesis in a sea of chemokine family members with a plethora of redundant activities. Exp. Hematol. 27:1113.[Medline]
  12. Zhou, Z., Y. J. Kim, K. Pollok, J. Hurtado, J. K. Lee, H. E. Broxmeyer, B. S. Kwon. 1993. Macrophage inflammatory protein-1{alpha} rapidly modulates its receptors and inhibits the anti-CD3 mAb-mediated proliferation of T lymphocytes. J. Immunol. 151:4333.[Abstract]
  13. Taub, D. D., S. M. Turcovski Corrales, M. L. Key, D. L. Longo, W. J. Murphy. 1996. Chemokines and T lymphocyte activation. I. {beta} chemokines costimulate human T lymphocyte activation in vitro. J. Immunol. 156:2095.[Abstract]
  14. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  15. Berg, E. L., T. Yoshino, L. S. Rott, M. K. Robinson, R. A. Warnock, T. K. Kishimoto, L. J. Picker, E. C. Butcher. 1991. The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1. J. Exp. Med. 174:1461.[Abstract/Free Full Text]
  16. Butcher, E. C., M. Williams, K. Youngman, L. Rott, M. Briskin. 1999. Lymphocyte trafficking and regional immunity. Adv. Immunol. 72:209.[Medline]
  17. Bargatze, R. F., M. A. Jutila, and E. C. Butcher. 1995. Distinct roles of L-selectin and integrins {alpha}4{beta}7 and LFA-1 in lymphocyte interactions with Peyer’s patch-HEV in situ: the multi-step hypothesis confirmed and refined. Immunity. In press.
  18. Campbell, J. J., J. Hedrick, A. Zlotnik, M. A. Siani, D. A. Thompson, E. C. Butcher. 1998. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279:381.[Abstract/Free Full Text]
  19. Kim, C. H., H. E. Broxmeyer. 1999. Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J. Leukocyte Biol. 65:6.[Abstract]
  20. Qin, S., J. B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, A. E. Koch, B. Moser, C. R. Mackay. 1998. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101:746.[Medline]
  21. Liao, F., R. L. Rabin, C. S. Smith, G. Sharma, T. B. Nutman, J. M. Farber. 1999. CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3{alpha}. J. Immunol. 162:186.[Abstract/Free Full Text]
  22. Qin, S., G. LaRosa, J. J. Campbell, H. Smith-Heath, N. Kassam, X. Shi, L. Zeng, E. C. Butcher, C. R. Mackay. 1996. Expression of monocyte chemoattractant protein-1 and interleukin-8 receptors on subsets of T cells: correlation with transendothelial chemotactic potential. Eur. J. Immunol. 26:640.[Medline]
  23. Adema, G. J., F. Hartgers, R. Verstraten, E. de Vries, G. Marland, S. Menon, J. Foster, Y. Xu, P. Nooyen, T. McClanahan, et al 1997. A dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells. Nature 387:713.[Medline]
  24. Andrew, D. P., M. S. Chang, J. McNinch, S. T. Wathen, M. Rihanek, J. Tseng, J. P. Spellberg, III C. G. Elias. 1998. STCP-1 (MDC) CC chemokine acts specifically on chronically activated Th2 lymphocytes and is produced by monocytes on stimulation with Th2 cytokines IL-4 and IL-13. J. Immunol. 161:5027.[Abstract/Free Full Text]
  25. Gonzalo, J. A., C. M. Lloyd, L. Kremer, E. Finger, A. C. Martinez, M. H. Siegelman, M. Cybulsky, J. C. Gutierrez-Ramos. 1996. Eosinophil recruitment to the lung in a murine model of allergic inflammation: the role of T cells, chemokines, and adhesion receptors. J. Clin. Invest. 98:2332.[Medline]
  26. Tedla, N., H. W. Wang, H. P. McNeil, N. Di Girolamo, T. Hampartzoumian, D. Wakefield, A. Lloyd. 1998. Regulation of T lymphocyte trafficking into lymph nodes during an immune response by the chemokines macrophage inflammatory protein (MIP)-1{alpha} and MIP-1{beta}. J. Immunol. 161:5663.[Abstract/Free Full Text]
  27. Carter, L. L., R. W. Dutton. 1996. Type 1 and type 2: a fundamental dichotomy for all T-cell subsets. Curr. Opin. Immunol. 8:336.[Medline]
  28. Mosmann, T. R., S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17:138.[Medline]
  29. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129.[Abstract/Free Full Text]
  30. 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]
  31. 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]
  32. Zingoni, A., H. Soto, J. A. Hedrick, A. Stoppacciaro, C. T. Storlazzi, F. Sinigaglia, D. D’Ambrosio, A. O’Garra, D. Robinson, M. Rocchi, et al 1998. The chemokine receptor CCR8 is preferentially expressed in Th2 but not Th1 cells. J. Immunol. 161:547.[Abstract/Free Full Text]
  33. Imai, T., M. Nagira, S. Takagi, M. Kakizaki, M. Nishimura, J. Wang, P. W. Gray, K. Matsushima, O. Yoshie. 1999. Selective recruitment of CCR4-bearing Th2 cells toward antigen-presenting cells by the CC chemokines thymus and activation-regulated chemokine and macrophage-derived chemokine. Int. Immunol. 11:81.[Abstract/Free Full Text]
  34. Luster, A. D., J. C. Unkeless, J. V. Ravetch. 1985. {gamma}-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315:672.[Medline]
  35. Bonecchi, R., S. Sozzani, J. T. Stine, W. Luini, G. D’Amico, P. Allavena, D. Chantry, A. Mantovani. 1998. Divergent effects of interleukin-4 and interferon-{gamma} on macrophage-derived chemokine production: an amplification circuit of polarized T helper 2 responses. Blood 92:2668.[Abstract/Free Full Text]
  36. Ponath, P. D., S. Qin, T. W. Post, J. Wang, L. Wu, N. P. Gerard, W. Newman, C. Gerard, C. R. Mackay. 1996. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J. Exp. Med. 183:2437.[Abstract/Free Full Text]
  37. Wu, L., N. Ruffing, X. Shi, W. Newman, D. Soler, C. R. Mackay, S. Qin. 1996. Discrete steps in binding and signaling of interleukin-8 with its receptor. J. Biol. Chem. 271:31202.[Abstract/Free Full Text]
  38. Wu, L., N. Gerard, R. Wyatt, H. Choe, C. Parolin, N. Ruffing, A. Borsetti, A. A. Cardoso, E. Desjardin, W. Newman, C. Gerard, J. Sodroski. 1996. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature 384:179.[Medline]
  39. Sornasse, T., P. V. Larenas, K. A. Davis, J. E. de Vries, H. Yssel. 1996. Differentiation and stability of T helper 1 and 2 cells derived from naive human neonatal CD4+ T cells, analyzed at the single-cell level. J. Exp. Med. 184:473.[Abstract/Free Full Text]
  40. Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober. 1992. Current Protocols in Immunology Wiley, New York.
  41. Campbell, J. J., G. Haraldsen, J. Pan, J. Rottman, S. Qin, P. Ponath, D. P. Andrew, R. Warnke, N. Ruffing, N. Kassam, et al 1999. The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells. Nature 400:776.[Medline]
  42. Bernardini, G., J. Hedrick, S. Sozzani, W. Luini, G. Spinetti, M. Weiss, S. Menon, A. Zlotnik, A. Mantovani, A. Santoni, M. Napolitano. 1998. Identification of the CC chemokines TARC and macrophage inflammatory protein-1{beta} as novel functional ligands for the CCR8 receptor. Eur. J. Immunol. 28:582.[Medline]
  43. Garlisi, C. G., H. Xiao, F. Tian, J. A. Hedrick, M. M. Billah, R. W. Egan, S. P. Umland. 1999. The assignment of chemokine-chemokine receptor pairs: TARC and MIP-1{beta} are not ligands for human CC-chemokine receptor 8. Eur. J. Immunol. 29:3210.[Medline]
  44. Chantry, D., P. Romagnani, C. J. Raport, C. L. Wood, A. Epp, S. Romagnani, P. W. Gray. 1999. Macrophage-derived chemokine is localized to thymic medullary epithelial cells and is a chemoattractant for CD3+, CD4+, CD8low thymocytes. Blood 94:1890.[Abstract/Free Full Text]
  45. Imai, T., T. Yoshida, M. Baba, M. Nishimura, M. Kakizaki, O. Yoshie. 1996. Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein-Barr virus vector. J. Biol. Chem. 271:21514.[Abstract/Free Full Text]
  46. Godiska, R., D. Chantry, C. J. Raport, S. Sozzani, P. Allavena, D. Leviten, A. Mantovani, P. W. Gray. 1997. Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells. J. Exp. Med. 185:1595.[Abstract/Free Full Text]
  47. Lieberam, I., I. Forster. 1999. The murine {beta}-chemokine TARC is expressed by subsets of dendritic cells and attracts primed CD4+ T cells. Eur. J. Immunol. 29:2684.[Medline]
  48. Akdis, M., C. A. Akdis, L. Weigl, R. Disch, K. Blaser. 1997. Skin-homing, CLA+ memory T cells are activated in atopic dermatitis and regulate IgE by an IL-13-dominated cytokine pattern: IgG4 counter-regulation by CLA-memory T cells. J. Immunol. 159:4611.[Abstract]
  49. Santamaria Babi, L. F., L. J. Picker, M. T. Perez Soler, K. Drzimalla, P. Flohr, K. Blaser, C. Hauser. 1995. Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen. J. Exp. Med. 181:1935.[Abstract/Free Full Text]
  50. Austrup, F., D. Vestweber, E. Borges, M. Lohning, R. Brauer, U. Herz, H. Renz, R. Hallmann, A. Scheffold, A. Radbruch, A. Hamann. 1997. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature 385:81.[Medline]
  51. Teraki, Y., L. J. Picker. 1997. Independent regulation of cutaneous lymphocyte-associated antigen expression and cytokine synthesis phenotype during human CD4+ memory T cell differentiation. J. Immunol. 159:6018.[Abstract]



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