HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sebastiani, S. Articles by Cavani, A. Search for Related Content PubMed PubMed Citation Articles by Sebastiani, S. Articles by Cavani, A.

Chemokine Receptor Expression and Function in CD4⁺ T Lymphocytes with Regulatory Activity¹

Silvia Sebastiani^*, Paola Allavena, Cristina Albanesi^*, Francesca Nasorri^*, Giancarlo Bianchi, Claudia Traidl^*, Silvano Sozzani, Giampiero Girolomoni^* and Andrea Cavani^2,*

^* Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy; and Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have investigated the chemokine receptor expression and migratory behavior of a new subset of nickel-specific skin-homing regulatory CD4⁺ T cells (Th^IL-10) releasing high levels of IL-10, low IFN-, and undetectable IL-4. These cells inhibit in a IL-10-dependent manner the capacity of dendritic cells to activate nickel-specific Tc1 and Th1 lymphocytes. RNase protection assay and FACS analysis revealed the expression of a vast repertoire of chemokine receptors on resting Th^IL-10, including the Th1-associated CXCR3 and CCR5, and the Th2-associated CCR3, CCR4, and CCR8, the latter at higher levels compared with Th2 cells. The most active chemokines for resting Th^IL-10, in terms of calcium mobilization and in vitro migration, were in order of potency: CCL2 (monocyte chemoattractant protein-1, CCR2 ligand), CCL4 (macrophage-inflammatory protein-1, CCR5 ligand), CCL3 (macrophage-inflammatory protein-1, CCR1/5 ligand), CCL17 (thymus and activation-regulated chemokine, CCR4 ligand), CCL1 (I-309, CCR8 ligand), CXCL12 (stromal-derived factor-1, CXCR4), and CCL11 (eotaxin, CCR3 ligand). Consistent with receptor expression down-regulation, activated Th^IL-10 exhibited a reduced or absent response to most chemokines, but retained a significant migratory capacity to I-309, monocyte chemoattractant protein-1, and thymus and activation-regulated chemokine. I-309, which was ineffective on Th1 lymphocytes, attracted more efficiently Th^IL-10 than Th2 cells. I-309 and CCR8 mRNAs were not detected in unaffected skin and were up-regulated at the skin site of nickel-allergic reaction, with an earlier expression kinetics compared with IL-10 and IL-4. Results indicate that skin-homing regulatory Th^IL-10 lymphocytes coexpress functional Th1- and Th2-associated chemokine receptors, and that CCR8/I-309-driven recruitment of both resting and activated Th^IL-10 cells may be critically involved in the regulation of Th1-mediated skin allergic disorders.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Compelling evidence indicates the existence of specialized CD4⁺ T lymphocytes with the potential to regulate a variety of immune responses through the release of suppressive cytokines (1, 2, 3). In particular, IL-10-producing CD4⁺ T cells appear to be profoundly involved in the modulation of autoimmune diseases as well as in the induction of transplantation tolerance (4, 5, 6). Allergic diseases are the consequence of exaggerated immune responses against innocuous non-self Ags. Allergic contact dermatitis to nickel is a prototypic type 1-mediated immune response and depends upon the recruitment of Th1 and Tc1 lymphocytes at the skin site of nickel application in sensitized individuals (7, 8, 9, 10). We have recently found that the skin and peripheral blood of nickel-allergic patients as well as the blood of healthy donors bear a substantial proportion of nickel-specific CD4⁺ T clones which produce high amounts of IL-10, low or undetectable IFN-, and no IL-4 (11). These nickel-specific IL-10-producing Th cells (Th^IL-10)³ express the skin-homing receptor, cutaneous lymphocyte-associated Ag (CLA), and block in a IL-10-dependent manner the differentiation and maturation of dendritic cells, thus impairing their capacity to activate nickel-specific Tc1 and Th1 effector lymphocytes (12).

Many recent findings indicate that chemokine receptors are differentially expressed on memory T cells depending on their polarization (13, 14, 15, 16, 17, 18), with Th1 lymphocytes expressing CCR5 and CXCR3, and Th2 lymphocytes CCR3, CCR4, and CCR8. As a consequence, selective chemotactic stimuli contribute to the differential positioning of Th1 and Th2 cells within tissues (19, 20, 21). Here, we analyzed the chemokine receptor profile and migratory properties of skin-homing nickel-specific Th^IL-10 lymphocytes. Our results demonstrate that resting Th^IL-10 cells coexpress functional Th1 and Th2 chemokine receptors and migrate in response to a variety of chemokines. Upon activation, Th^IL-10 cells are attracted exclusively by CCL1 (I-309), CCL2 (monocyte chemoattractant protein-1, MCP-1), CCL17 (thymus and activation-regulated chemokine, TARC), and CXCL12 (stromal derived factor 1, SDF-1). I-309, which is produced in the skin affected with allergic contact dermatitis to nickel, attracts Th^IL-10 and Th2, but not Th1 cells, and may thus contribute to the regulation of the Th1-mediated skin inflammation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Culture medium and Abs

T lymphocytes were cultured in RPMI 1640 supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 0.05 mM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Chagrin Falls, OH) (complete RPMI) plus 10% heat-inactivated FCS (HyClone Logan, UT) or 5% autologous plasma. The anti-CD28 mAb (Leu-28, IgG1) was purchased from Becton Dickinson (San Jose, Ca). Mouse mAbs anti-human CCR1 (53504.111, IgG2_B), CCR2 (48607.121, IgG2_B), CCR5 (45531.111, IgG2_B), and CXCR3 (49801.111, IgG1) were obtained from R&D Systems (Minneapolis, MN); anti-CCR3 (LS63 7B11, IgG2_A) and anti-CCR4 (328B, IgG) mAbs were kindly provided by Leukosite (Cambridge, MA) and ICOS (Bothell, WA), respectively; anti-CXCR1 (5A12, IgG2_B), CXCR2 (6C6, IgG1) and CXCR4 (12G5, IgG2_A) were purchased from PharMingen (San Diego, CA). Anti-CD3 (UCHT-1, IgG1) was obtained from Immunotech (Marseille, France); the FITC-conjugated anti-CLA mAb (HECA-452, rat IgM), the control isotype FITC-conjugated rat IgM, and the control mouse IgG were purchased from PharMingen. FITC-conjugated goat anti-mouse IgG were obtained from Dako (Glostrup, Denmark).

T cell clones

T cell lines were derived from the blood and skin of nonatopic patients affected by allergic contact dermatitis to nickel, as previously described (12). Briefly, >95% pure CD4⁺ T cells were negatively selected from the nonadherent fraction of PBMC using immunomagnetic beads coated with anti-CD19, anti-HLA-DR, and anti-CD8 mAbs (Dynal, Oslo, Norway). Blood-derived CD4⁺ T cells were expanded with autologous PBMC and 10 µg/ml NiSO₄ (Sigma-Aldrich, Milan, Italy) to enrich for nickel-specific T cells. Skin-derived T cells were obtained from 4-mm punch biopsies performed 48 h after the application of 5% NiSO₄ in petrolatum on the back of allergic patients. After extensive washing in PBS, biopsies were placed in culture at 37°C with 5% CO₂ in complete RPMI along with 5% autologous plasma and 30 U/ml rhIL-2 (generously provided by Chiron Italia, Milan, Italy). Medium was replaced every third day, and T cells emigrating from the tissue were collected at day 10. Blood- and skin-derived T cell lines were cloned by limiting dilution (0.5 cells/well) in the presence of 2 x 10⁵ PBMC, 30 U/ml IL-2, and 1% PHA in complete RPMI plus 10% FCS. Clones were grown in the presence of IL-2 and periodically stimulated with 1% PHA and feeder cells or plate-coated anti-CD3 (1 µg/ml) and soluble anti-CD28 (1 µg/ml) mAbs. Ag specificity of T cell lines and clones was tested using irradiated PBMC and 10 µg/ml NiSO₄ in complete RPMI supplemented with 5% autologous plasma for 48 h and pulsed with 5 µCi/ml [³H]TdR (Amersham, Little Chalfont, U.K.) for the last 12 h of culture. Cultures were harvested onto fiber-coated 96-well plates, and thymidine incorporation was measured in a beta counter (Topcount; Packard, Groningen, The Netherlands). Supernatants from T cells (10⁶ cells/ml) stimulated in 24-well plates with immobilized anti-CD3 and soluble anti-CD28 mAbs were collected after 48 h, filtered, and tested for IL-10, IL-4, IFN-, and TGF- content by ELISA (R&D Systems).

RNase protection assay

Total RNA was extracted from resting and 6-to 48-h activated T cell clones using TRIzol (Life Technologies) according to the manufacturer’s instructions. Two multiprobe template sets, hCR5 and hCR6 (RiboQuant; PharMingen), were used for in vitro transcription reactions in the presence of a GACU pool and a T7 RNA polymerase to synthesize [³²P]UTP-labeled antisense probes. RNase protection analysis of 2.5 µg of total RNA was performed after overnight hybridization at 60°C with 2.5 x 10⁶ cpm of hCR5 or hCR6, followed by digestion with RNase A and T1 according to standard protocols. Protected fragments were treated with proteinase K, extracted with phenol-chloroform plus isoamyl alcohol (50:1), and finally precipitated in ethanol in the presence of ammonium acetate. The samples were electrophoresed on 5% denaturing sequencing gels and then exposed on film (Kodak, Rochester, NY). The levels of mRNA expression were quantified by densitometric analysis with an imaging densitometer (model GS-670; Bio-Rad, Hercules, CA), supported by the Molecular Analyst Image software, and the values were normalized against the housekeeping gene L32.

FACS analysis

Th^IL-10 lymphocytes in resting condition or activated 48 h with immobilized anti-CD3 were washed in PBS added with 1% FBS and stained with specific mAb or isotype control Ig, followed by secondary anti-mouse FITC-conjugated Ab. For CCR1 detection, cells were preincubated for 15 min with 2% paraformaldehyde and then permeabilized with 0.5% saponin for 20 min before staining with the mAb or the isotype control. Cells were analyzed with a FACScan equipped with CellQuest software (Becton Dickinson).

Intracellular Ca²⁺ concentration measurements

T cell clones were either left untreated or activated with anti-CD3 plus anti-CD28 for 48 h in the presence of 30 U/ml IL-2. Thereafter, cells (5 x 10⁶/ml) were washed and loaded with 8 µM Fluo-3/acetoxymethyl ester in the presence of 1 µM pluronic F-127 (Molecular Probes, Eugene, OR) in complete RPMI with 1% FBS for 40 min at 37°C with frequent gentle agitation. Cells were then washed twice, stimulated with CCL3 (macrophage inflammatory protein (MIP)-1, 200 ng/ml), MCP-1 (200 ng/ml), CCL11 (eotaxin, 400 ng/ml), TARC (100 ng/ml), CCL4 (MIP-1, 100 ng/ml), CCL19 (MIP-3, 30 ng/ml), I-309 (200 ng/ml), CXCL8 (IL-8, 100 ng/ml), CXCL10 (IFN-induced protein of 10 kDa (IP-10), 100 ng/ml), and SDF-1 (100 ng/ml) (all from R&D Systems), and finally analyzed on a FACScan. Cells and chemokines were maintained at 37°C during the assay.

Migration assay

Chemotactic property of each chemokine was evaluated measuring the lymphocyte migration through a 5-µm pore polycarbonate filter in 24-well transwell chambers (Corning Costar, Cambridge MA) as previously described (22). Briefly Th1, Th2, and Th^IL-10 either in resting or stimulated 48 h with immobilized anti-CD3 plus anti-CD28 were added to the top chamber suspended in complete RPMI plus 0.5% BSA at 1 x 10⁶ cells/ml. Various concentrations of the different chemokines were added to the bottom chamber of the transwell (0.6 ml). After 1 h of incubation at 37°C with 5% CO₂, cells transmigrated into the lower chamber were recovered and counted with a FACScan for 60 s at a flow rate of 60 µl/min, as previously described (23). Results are shown as migration index, which represents the ratio between T cells migrated to the lower chamber in the presence of the agonistic chemokine and cells migrated in response to the medium alone.

RT-PCR analysis

Total cellular RNA was extracted from 4-mm punch skin biopsies obtained at time 0 and 16, 48 and 60 h after the application of 5% NiSO₄ on the back of allergic individuals (n = 2) using the acid guanidinium thiocyanate-phenol-chloroform method. Total RNA (0.5–1 µg) was reverse transcribed using oligo(dT) primers and then subjected to amplification with a GeneAmp RNA PCR kit (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ). The following synthetic oligonucleotides were used: for I-309, 5'-ATG CAG ATC ATC ACC ACA GCC CTG and 3'-ACA GAA AAA TGC TGA GGC ACT GCC (274-bp amplificate); for CCR8, 5'-CAT CAC CCT CAT GAG TGT GG and 3'-CAC GTT GAA TGG GAC CCA GA (410-bp amplificate); for IL-10, 5'-GAA GGA TCA GCT GGA CAA CTT GTT G and 3'-GCT CCA AAT GTA GGG GCA GG (306-bp amplificate); for IL-4, 5'-GCG ATA TCA CCT TAC AGG AG and 3'-TTG GCT TCC TTC ACA GGA CA (308-bp amplificate); for IFN-, 5'-TGC AGG TCA TTC AGA TGT AG and 3'-AGC CAT CAC TTG GAT GAG TT (306-bp amplificate) (24). The -actin housekeeping gene was amplified with 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA and 3'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG primers (660-bp amplificate). To eliminate genomic DNA contamination, reverse transcription was performed after digestion with RNase-free DNase (PharMingen) for 30 min at 37°C. As a control, reactions were also performed in the absence of reverse transcriptase. For semiquantitative analysis, RNA concentrations, primers, and PCR cycles were titrated to obtain standard curves to verify linearity and to permit analysis of signal strength. The levels of mRNA expression were quantified by densitometric analysis as described above, with the values normalized against -actin.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chemokine receptor mRNA in resting and activated Th1, Th2, and Th^IL-10 nickel-specific T cells clones

T cell clones were isolated from either the peripheral blood or lesional skin of patients affected with allergic contact dermatitis to nickel. Th^IL-10 clones released high amounts of IL-10, low levels of IFN-, and very limited amounts or no IL-4, and expressed the CLA receptor which allows the recruitment of memory T cells in the skin (8) (Table I). To determine the chemokine receptor expression, T cell clones were collected in resting phase and 6–48 h after anti-CD3/anti-CD28-induced activation, and the mRNA content was evaluated by RNase protection assay using CC and CXC chemokine receptor multiprobe template sets. In resting conditions, Th^IL-10 cells expressed a wide range of CC and CXC receptors (Figs. 1 and 2). As previously reported for other T cell subsets (25), TCR engagement strongly affected chemokine receptor expression. After a transient burst at 6 h in CCR1, CCR2, CCR4, CCR5, and CCR8 mRNA, Th^IL-10 clones down-regulated CXC (CXCR1, CXCR3, and CXCR4) receptors, CCR2, and, to a lesser extent, CCR3 and CCR8. In contrast, CCR1, CCR4, CCR5, and CCR7 were up-regulated. When compared with Th1 and Th2, Th^IL-10 lymphocytes displayed a broader array of chemokine receptors, with significant expression of both the Th1-associated CCR5 and CXCR3 and the Th2-associated CCR3, CCR4, and CCR8 (13, 14, 15, 16, 17, 18, 25). Interestingly, the mRNA signal for CCR8 was 2-fold stronger in resting Th^IL-10 lymphocytes than in Th2 clones, and the difference was even more pronounced upon activation, being CCR8 markedly down-regulated in TCR-engaged Th2 lymphocytes, but only slightly in Th^IL-10 cells. Th^IL-10, but not Th1 and Th2, clones expressed low levels of CCR7 mRNA, which was up-regulated upon activation. Finally, both resting and stimulated Th^IL-10 clones showed 1.5- to 3.5-fold higher CXCR4 mRNA levels than Th1 and Th2 cells.

View larger version (64K): [in this window] [in a new window]   FIGURE 1. CC and CXC chemokine receptor mRNA expressed by nickel-specific ThIL-10, Th1, and Th2 clones. Receptor mRNAs were examined by RNase protection assay in resting T cell clones and 6–48 h after stimulation with plate-bound anti-CD3 and soluble anti-CD28 in the presence of exogenous IL-2 (30 U/ml). Similar results were observed in four ThIL-10, two Th1, and two Th2 clones.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Densitometric analysis of chemokine receptor mRNAs expressed by resting and activated T cell clones. Densitometric values were calculated from RNase protection assay by laser densitometry and normalized against the housekeeping gene L32. Results are expressed as the mean ± SD of experiments performed on four ThIL-10, two Th1, and two Th2 clones. Open columns, resting T cells; hatched columns, 6-h activated T cells; and filled columns, 48-h activated T cells.

FACS analysis confirmed the expression of the CCRs 1–5 and the CXCRs 1, 3, and 4 on resting Th^IL-10 lymphocytes, whereas CXCR2 was negative (Fig. 3). Activated Th^IL-10 lymphocytes strongly reduced the surface expression of CCR1, CCR3, CCR5, CXCR1, and CXCR3. Although CCR1 and CCR5 protein expression decreased, the corresponding mRNA signals were up-regulated following TCR triggering. The expression of CCR2 and CCR4, albeit reduced, was maintained at significant levels and that of CXCR4 was unmodified after Th^IL-10 activation.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Chemokine receptors expressed by resting and activated ThIL-10 lymphocytes. Cells in resting state (bold lines) or 48-h activated with immobilized anti-CD3 (thin lines) were stained with the specific mAbs or with the isotype control Ig (dashed lines), followed by FITC-conjugated secondary Ab, and then examined with a FACScan. Results are representative of four ThIL-10 clones.

To evaluate the functional relevance of chemokine receptor expression, we measured calcium mobilization in Th^IL-10 lymphocytes after treatment with agonistic chemokines. Consistent with the vast repertoire of receptors expressed, all of the chemokines tested induced significant responses in resting Th^IL-10 cells (Fig. 4). The most active chemokines were MCP-1 (CCR2 ligand), I-309 (CCR8 ligand), MIP-1 (CCR1–CCR5 ligand), and MIP-1 (CCR5 ligand), followed by TARC (CCR4 ligand), IL-8 (CXCR1 ligand) and eotaxin (CCR3 ligand), SDF-1 (CXCR4 ligand), and finally MIP-3 (CCR7 ligand). After activation, significant calcium fluxes were measured in response to I-309, MCP-1, TARC, and SDF-1, whereas the responses to the other chemokines were strongly reduced or completely lost. Consistent with the up-regulation of CCR7 mRNA, MIP-3 retained a slight stimulatory effect on activated Th^IL-10 clones. Notably, activated Th^IL-10 cells became unresponsive to MIP-1 and MIP-1, in line with the decreased expression of CCR1 and CCR5 receptors. This finding has been previously observed in mature dendritic cells and activated monocytes (26, 27).

View larger version (50K): [in this window] [in a new window]   FIGURE 4. Intracellular calcium fluxes induced in ThIL-10 clones by chemokines. ThIL-10 cells were loaded with 8 µM fluo-3/acetoxymethyl ester in the presence of 1 µM pluronic F-127 and tested for intracellular calcium mobilization in response to different concentrations of agonistic chemokines. Calcium fluxes were measured in the resting state and 48 h after activation with anti-CD3/anti-CD28 in the presence of 30 U/ml IL-2. Shown is one experiment of four performed with different ThIL-10 clones.

The migratory behavior of Th1, Th2, and Th^IL-10 lymphocytes was compared by measuring their transmigration through transwell filters in response to different concentrations of agonistic chemokines (Fig. 5). Resting Th^IL-10 revealed an extraordinary capacity to migrate in response to a broad array of chemotactic stimuli, including both Th1- and Th2-associated chemokines. The highest migration was achieved with MCP-1, MIP-1, and MIP-1, followed by TARC, I-309, SDF-1, and eotaxin, whereas more limited responses were observed to IL-8, IP-10, and MIP-3. Th^IL-10 migration to MIP-1, IP-10 (CCR5 and CXCR3 ligands), and to eotaxin (CCR3 ligand) was lower compared with that of Th1 and Th2 clones, respectively. In contrast, I-309 induced a stronger migration in Th^IL-10 than in Th2 cells. Responses to MIP-1, MCP-1, and TARC were comparable among the different Th cell subsets, whereas Th^IL-10 and Th2 migrated to a higher extent in response to IL-8 compared with Th1 clones. IL-8 responsiveness was likely due to CXCR1, but not CXCR2 triggering, since the latter receptor was undetectable on Th^IL-10 both at mRNA and protein levels. Additionally, neutrophil-activating protein-2, a specific agonist of CXCR2 but not CXCR1, failed to induce calcium mobilization and migration of Th^IL-10 (data not shown). Finally, only Th^IL-10 cells showed a slight migration to MIP-3. After stimulation with anti-CD3 and anti-CD28 mAbs, Th^IL-10 cells markedly decreased their migratory response to most of the chemokines, and, consistent with the calcium fluxes, retained a significant migration to MCP-1, I-309, TARC, and SDF-1. MCP-1 and TARC were also effective in attracting activated Th1 and Th2 lymphocytes, whereas I-309 attracted Th2, but not Th1 cells. Activated Th1 lymphocytes maintained a significant response to MIP-1, MIP-1, and IP-10.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Migratory response of ThIL-10 cells to chemokines. Migratory responses to various concentrations of CC and CXC chemokines was examined in resting () and activated () ThIL-10 cells. Data are expressed as migration index (number of cells migrated with the agonistic chemokine/number of cells migrated with medium alone; mean ± SD). Similar results were obtained in three separate experiments using different ThIL-10 clones.

Since I-309 appeared to selectively attract Th^IL-10 and Th2 lymphocytes and was effective on both resting and activated Th^IL-10 cells, its expression was investigated during allergic contact dermatitis reaction, in combination with CCR8, IL-10, IL-4, and IFN-. RT-PCR was performed on RNA extracted from unaffected (time 0) skin and at 16, 48, and 60 h after nickel challenge. Fig. 6 demonstrates that I-309, CCR8, IL-10, and IL-4 mRNAs were undetectable in normal skin. Both CCR8 and I-309 were already expressed at 16 h after nickel application and reached a peak at 48 h, whereas IL-4 and IL-10 mRNAs became detectable only after 48 h. In contrast, a faint signal for IFN- mRNA was already present in unaffected skin, augmented at 16–48 h, and decreased thereafter.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. I-309, CCR8, and cytokine mRNA expression during allergic contact dermatitis to nickel. A, RT-PCR analysis was performed on RNA extracted from skin biopsies obtained from nickel-allergic patients at time 0 and at 16–60 h after the application of 5% NiSO4 in petroleum. B, Densitometric analysis was performed by normalizing the levels of RNA against -actin. Similar results were obtained in biopsies from two patients.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-10-releasing T lymphocytes with regulatory function have been obtained in vivo after repeated intranasal stimulation with peptide Ag (29) or repeated superantigen challenge (30), and following immunization through UVB-irradiated skin (31). In addition, Groux et al. (32) have shown that murine and human lymphocytes cultured in the presence of IL-10 develop a phenotype characterized by high release of IL-10, IL-5, and TGF-, variable amounts of IFN-, and no IL-4. These lymphocytes, called T regulatory cells 1 (Tr1), inhibit immune responses both in vitro and in vivo in a IL-10-dependent manner. More recently, a Tr1 polarization was induced in vitro by using dendritic cells treated with corticosteroids (33). Our skin-homing Th^IL-10 cells resemble Tr1 lymphocytes, but release lower levels of IFN- and display variable TGF- production. Moreover, nickel-specific Th^IL-10 lymphocytes display the IL-12R 2 chain, the CD26 and the lymphocyte activation Ag-3 (12), markers related to a Th1 polarization. Whether these suppressive Th^IL-10 cells represent a distinct Th subset or a further maturation step of Th1 or Th2 cells has to be determined.

An interesting observation of our study was that I-309 was capable of attracting more efficiently Th^IL-10 than Th2 cells, both in resting and activated conditions, whereas it was ineffective on Th1. We also found that I-309 mRNA was significantly expressed in the skin after Ag challenge. I-309 expression paralleled that of CCR8 and preceded IL-4 and IL-10 expression. I-309-mediated recruitment of Th^IL-10 cells may thus represent an important mechanism for terminating Th1-mediated allergic reactions and limiting excessive tissue damage. In line with this hypothesis, it has been shown that administration of IL-10 strongly reduces murine contact hypersensitivity (34, 35), and IL-10-deficient mice show enhanced and more sustained cutaneous inflammatory responses to haptens (36). Activated Th1 and Th2 lymphocytes are the major source of I-309 (25, 37), but we have recently found a significant production of I-309 by IFN--stimulated keratinocytes (C. Albanesi, C. Scarponi, S. Sebastiani, A. Cavani, M. Federici, S. Sozzani, and G. Girolomoni, manuscript in preparation), which may therefore contribute relevantly to the recruitment of regulatory Th^IL-10 cells in the skin. In aggregate, our results demonstrate that Th^IL-10 display a broad array of functional chemokine receptors, which allow their comigration along with Th1 and Th2 cells. The expression of high levels of CCR8 in regulatory Th^IL-10 lymphocytes may be advantageously exploited to control Th1-mediated allergic disorders.

	Acknowledgments

 
We would thank Dr. Monica Napolitano for the CCR8 primers and Dr. Alberto Mantovani for critical review of this manuscript.

	Footnotes

 
¹ This work was supported by the European Community (Biomed 2 Program, Grant BMH4-CT98h3713), the Istituto Superiore di Sanità (AIDS Project, Grant 40B/1.18), the Associazione Italiana per la Ricerca sul Cancro, and the Ministero della Sanità.

² Address correspondence and reprint requests to Dr. Andrea Cavani, Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS Via Monti di Creta 104, I-00167 Rome, Italy.

³ Abbreviations used in this paper: Th^IL-10, IL-10-producing Th cells; CLA, cutaneous lymphocyte-associated Ag; MCP, monocyte chemoattractant protein; TARC, thymus and activation-regulated chemokine; SDF, stromal-derived factor; MIP, macrophage-inflammatory protein; IP-10, IFN-induced protein of 10 kDa; Tr1, T regulatory cell 1.

Received for publication July 27, 2000. Accepted for publication October 30, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. O’Garra, A., L. Steinman, K. Gijbels. 1997. CD4⁺ T-cell subsets in autoimmunity. Curr. Opin. Immunol. 9:872.[Medline]
2. Mason, D., F. Powrie. 1998. Control of immune pathology by regulatory T cells. Curr. Opin. Immunol. 10:649.[Medline]
3. Shevach, E. M.. 2000. Regulatory T cells in autoimmunity. Annu. Rev. Immunol. 18:423.[Medline]
4. Stohlman, S. A., L. Pei, D. J. Cua, Z. Li, D. R. Hinton. 1999. Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10. J. Immunol. 163:6338.[Abstract/Free Full Text]
5. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory cells that inhibit intestinal inflammation. J. Exp. Med. 190:995.[Abstract/Free Full Text]
6. Zhai, Y., J. W. Kupiec-Weglinski. 1999. What is the role of regulatory T cells in transplantation tolerance?. Curr. Opin. Immunol. 11:497.[Medline]
7. Sinigaglia, F.. 1994. The molecular basis of metal recognition by T cells. J. Invest. Dermatol. 102:398.[Medline]
8. Robert, V., T. S. Kupper. 1999. Inflammatory skin diseases, T cells, and immune surveillance. N. Engl. J. Med. 341:1817.[Free Full Text]
9. Albanesi, C., C. Scarponi, S. Sebastiani, M. Federici, A. Cavani, O. De Pità, P. Puddu, G. Girolomoni. 2000. IL-4 enhances keratinocyte production of CXCR3 agonistic chemokines. J. Immunol. 165:1395.[Abstract/Free Full Text]
10. Traidl, C., S. Sebastiani, C. Albanesi, H. F. Merk, P. Puddu, G. Girolomoni, A. Cavani. 2000. Disparate cytotoxic activity of nickel-specific CD8⁺ and CD4⁺ T cell subsets against keratinocytes. J. Immunol. 165:3058.[Abstract/Free Full Text]
11. Cavani, A., D. Mei, E. Guerra, S. Corinti, M. Giani, L. Pirrotta, P. Puddu, G. Girolomoni. 1998. Patients with allergic contact dermatitis to nickel and nonallergic individuals display different nickel-specific T cell responses: evidence for the presence of effector CD8⁺ and regulatory CD4⁺ T cells. J. Invest. Dermatol. 111:621.[Medline]
12. Cavani, A., F. Nasorri, C. Prezzi, S. Sebastiani, C. Albanesi, G. Girolomoni. 2000. Human CD4⁺ T lymphocytes with remarkable regulatory functions on dendritic cells and Th1 immune responses. J. Invest. Dermatol. 114:295.[Medline]
13. 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]
14. Gerber, B. O., M. P. Zanni, M. Uguccioni, M. Loetscher, C. R. Mackay, W. J. Pichler, N. Yawalkar, M. Baggiolini, B. Moser. 1997. Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils. Curr. Biol. 7:836.[Medline]
15. 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]
16. 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]
17. Zingoni, A., H. Soto, J. A. Hedrick, A. Stoppacciaro, C. T. Storlazzi, F. Sinigaglia, D. D’Ambrosio, A. O’Garra, D. Robinson, M. Rocchi, A. Santoni, A. Zlotnik, M. Napolitano. 1998. The chemokine receptor CCR8 is preferentially expressed in Th2 but not in Th1 cells. J. Immunol. 161:547.[Abstract/Free Full Text]
18. D’Ambrosio, D., A. Iellem, R. Bonecchi, D. Mazzeo, S. Sozzani, A. Mantovani, F. Sinigaglia. 1998. Selective up-regulation of chemokine receptors CCR4 and CCR8 upon activation of polarized human type 2 Th cells. J. Immunol. 161:5111.[Abstract/Free Full Text]
19. Mantovani, A.. 1999. The chemokine system: redundancy for robust outputs. Immunol. Today 20:254.[Medline]
20. Baggiolini, M.. 1998. Chemokines and leukocyte traffic. Nature 392:565.[Medline]
21. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 2000. The role of chemokine receptors in primary, effector and memory immune responses. Annu. Rev. Immunol. 18:593.[Medline]
22. Colantonio, L., A. Iellem, B. Clissi, R. Pardi, L. Rogge, F. Sinigaglia, D. D’Ambrosio. 1999. Upregulation of integrin ₆₁ and chemokine receptor CCR1 by interleukin-12 promotes the migration of human type 1 helper T cells. Blood 94:2981.[Abstract/Free Full Text]
23. Bleul, C. C., R. C. Fuhlbrigge, J. M. Casanovas, A. Aiuti, T. A. Springer. 1996. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J. Exp. Med. 184:1101.[Abstract/Free Full Text]
24. Miller, M. D., S. D. Wilson, M. E. Dorf, H. N. Seuanez, S. J. O’Brien, M. S. Krangel. 1990. Sequence and chromosomal location of the I-309 gene: relationship to genes encoding a family of inflammatory cytokines. J. Immunol. 145:2737.[Abstract]
25. Sallusto, F., E. Kremmer, B. Palermo, A. Hoy, P. Ponath, S. Qin, R. Förster, M. Lipp, A. Lanzavecchia. 1999. Switch in chemokine receptor expression upon TCR stimulation reveals novel homing potential for recently activated T cells. Eur. J. Immunol. 29:2037.[Medline]
26. Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, S. Qin, A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur. J. Immunol. 28:2760.[Medline]
27. Franchin, G., G. Zybarth, W. W. Dai, L. Dubrovski, N. Reiling, H. Scmidtmayerova, M. Bukrinsky, B. Sherry. 2000. Lipopolysaccharide inhibits HIV-1 infection of monocyte-derived macrophages through direct and sustained down-regulation of CC chemokine receptor 5. J. Immunol. 164:2592.[Abstract/Free Full Text]
28. 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]
29. Burkhart, C., G. Y. Liu, S. M. Anderton, B. Metzler, D. C. Wraith. 1999. Peptide-induced T cell regulation of experimental autoimmune encephalomyelitis: a role for IL-10. Int. Immunol. 11:1625.[Abstract/Free Full Text]
30. Sundstedt, A., I. Hoiden, A. Rosendahl, T. Kalland, N. von Rooijen, M. Dohlsten. 1997. Immunoregulatory role of IL-10 during superantigen-induced hyporesponsiveness in vivo. J. Immunol. 158:180.[Abstract]
31. Shreedhar, V. K., M. W. Pride, Y. Sun, M. L. Kripke, F. M. Strickland. 1998. Origin and characteristics of ultraviolet-B radiation-induced suppressor T lymphocytes. J. Immunol. 161:1327.[Abstract/Free Full Text]
32. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, A. Svetlana, 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]
33. Matyszak, M. K., S. Citterio, M. Rescigno, P. Ricciardi-Castagnoli. 2000. Differential effects of corticosteroids during different stages of dendritic cell maturation. Eur. J. Immunol. 30:1233.[Medline]
34. Enk, A. H., J. Saloga, D. Becker, M. Mohamadzadeh, J. Knop. 1994. Induction of hapten-specific tolerance by interleukin 10 in vivo. J. Exp. Med. 179:1397.[Abstract/Free Full Text]
35. Ferguson, T. A., P. Dube, T. S. Griffith. 1994. Regulation of contact hypersensitivity by interleukin 10. J. Exp. Med. 179:1597.[Abstract/Free Full Text]
36. Berg, D. J., M. W. Leach, R. Kühn, K. Rajewsky, W. Müller, N. J. Davidson, D. Rennick. 1995. Interleukin 10 but not interleukin 4 is a natural suppressant of cutaneous inflammatory responses. J. Exp. Med. 182:99.[Abstract/Free Full Text]
37. Roos, R. S., M. Loetscher, D. F. Legler, I. Clark-Lewis, M. Baggiolini, B. Moser. 1997. Identification of CCR8, the receptor for the human CC chemokine I-309. J. Biol. Chem. 272:1725.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. E. Moreno-Fernandez, W. Zapata, J. T. Blackard, G. Franchini, and C. A. Chougnet Human Regulatory T Cells Are Targets for Human Immunodeficiency Virus (HIV) Infection, and Their Susceptibility Differs Depending on the HIV Type 1 Strain J. Virol., December 15, 2009; 83(24): 12925 - 12933. [Abstract] [Full Text] [PDF]

				A. Yessoufou, A. Ple, K. Moutairou, A. Hichami, and N. A. Khan Docosahexaenoic acid reduces suppressive and migratory functions of CD4CD25 regulatory T-cells J. Lipid Res., December 1, 2009; 50(12): 2377 - 2388. [Abstract] [Full Text] [PDF]

				S. Kimpfler, A. Sevko, S. Ring, C. Falk, W. Osen, K. Frank, M. Kato, K. Mahnke, D. Schadendorf, and V. Umansky Skin Melanoma Development in ret Transgenic Mice Despite the Depletion of CD25+Foxp3+ Regulatory T Cells in Lymphoid Organs J. Immunol., November 15, 2009; 183(10): 6330 - 6337. [Abstract] [Full Text] [PDF]

				M. C. Picchio, E. Scala, D. Pomponi, E. Caprini, M. Frontani, I. Angelucci, A. Mangoni, C. Lazzeri, M. Perez, D. Remotti, et al. CXCL13 Is Highly Produced by Sezary Cells and Enhances Their Migratory Ability via a Synergistic Mechanism Involving CCL19 and CCL21 Chemokines Cancer Res., September 1, 2008; 68(17): 7137 - 7146. [Abstract] [Full Text] [PDF]

				E. Mira, B. Leon, D. F. Barber, S. Jimenez-Baranda, I. Goya, L. Almonacid, G. Marquez, A. Zaballos, C. Martinez-A., J. V. Stein, et al. Statins Induce Regulatory T Cell Recruitment via a CCL1 Dependent Pathway J. Immunol., September 1, 2008; 181(5): 3524 - 3534. [Abstract] [Full Text] [PDF]

				D. Baatar, P. Olkhanud, D. Newton, K. Sumitomo, and A. Biragyn CCR4-Expressing T Cell Tumors Can Be Specifically Controlled via Delivery of Toxins to Chemokine Receptors J. Immunol., August 1, 2007; 179(3): 1996 - 2004. [Abstract] [Full Text] [PDF]

				D. Baatar, P. Olkhanud, K. Sumitomo, D. Taub, R. Gress, and A. Biragyn Human Peripheral Blood T Regulatory Cells (Tregs), Functionally Primed CCR4+ Tregs and Unprimed CCR4- Tregs, Regulate Effector T Cells Using FasL J. Immunol., April 15, 2007; 178(8): 4891 - 4900. [Abstract] [Full Text] [PDF]

				M. Kallikourdis, K. G. Andersen, K. A. Welch, and A. G. Betz Alloantigen-enhanced accumulation of CCR5+ 'effector' regulatory T cells in the gravid uterus PNAS, January 9, 2007; 104(2): 594 - 599. [Abstract] [Full Text] [PDF]

				A.-H. Zarnani, S.-M. Moazzeni, F. Shokri, M. Salehnia, and M. Jeddi-Tehrani Kinetics of murine decidual dendritic cells Reproduction, January 1, 2007; 133(1): 275 - 283. [Abstract] [Full Text] [PDF]

				C Lopez, M Comabella, M Tintore, J Sastre-Garriga, and X Montalban Variations in chemokine receptor and cytokine expression during pregnancy in multiple sclerosis patients Multiple Sclerosis, August 1, 2006; 12(4): 421 - 427. [Abstract] [PDF]

				M. Sironi, F. O. Martinez, D. D'Ambrosio, M. Gattorno, N. Polentarutti, M. Locati, A. Gregorio, A. Iellem, M. A. Cassatella, J. Van Damme, et al. Differential regulation of chemokine production by Fc{gamma} receptor engagement in human monocytes: association of CCL1 with a distinct form of M2 monocyte activation (M2b, Type 2) J. Leukoc. Biol., August 1, 2006; 80(2): 342 - 349. [Abstract] [Full Text] [PDF]

				M. G. Narducci, E. Scala, A. Bresin, E. Caprini, M. C. Picchio, D. Remotti, G. Ragone, F. Nasorri, M. Frontani, D. Arcelli, et al. Skin homing of Sezary cells involves SDF-1-CXCR4 signaling and down-regulation of CD26/dipeptidylpeptidase IV Blood, February 1, 2006; 107(3): 1108 - 1115. [Abstract] [Full Text] [PDF]

				N. Bendriss-Vermare, S. Burg, H. Kanzler, L. Chaperot, T. Duhen, O. de Bouteiller, M. D'agostini, J.-M. Bridon, I. Durand, J. M. Sederstrom, et al. Virus overrides the propensity of human CD40L-activated plasmacytoid dendritic cells to produce Th2 mediators through synergistic induction of IFN-{gamma} and Th1 chemokine production J. Leukoc. Biol., October 1, 2005; 78(4): 954 - 966. [Abstract] [Full Text] [PDF]

				N. R. Veillard, S. Steffens, G. Pelli, B. Lu, B. R. Kwak, C. Gerard, I. F. Charo, and F. Mach Differential Influence of Chemokine Receptors CCR2 and CXCR3 in Development of Atherosclerosis In Vivo Circulation, August 9, 2005; 112(6): 870 - 878. [Abstract] [Full Text] [PDF]

				C. M. Freeman, B.-C. Chiu, V. R. Stolberg, J. Hu, K. Zeibecoglou, N. W. Lukacs, S. A. Lira, S. L. Kunkel, and S. W. Chensue CCR8 Is Expressed by Antigen-Elicited, IL-10-Producing CD4+CD25+ T Cells, Which Regulate Th2-Mediated Granuloma Formation in Mice J. Immunol., February 15, 2005; 174(4): 1962 - 1970. [Abstract] [Full Text] [PDF]

				N. R. Veillard, S. Steffens, F. Burger, G. Pelli, and F. Mach Differential Expression Patterns of Proinflammatory and Antiinflammatory Mediators During Atherogenesis in Mice Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2339 - 2344. [Abstract] [Full Text] [PDF]

				J. Kipnis, M. Cardon, H. Avidan, G. M. Lewitus, S. Mordechay, A. Rolls, Y. Shani, and M. Schwartz Dopamine, through the Extracellular Signal-Regulated Kinase Pathway, Downregulates CD4+CD25+ Regulatory T-Cell Activity: Implications for Neurodegeneration J. Neurosci., July 7, 2004; 24(27): 6133 - 6143. [Abstract] [Full Text] [PDF]

				P. Schaerli, L. Ebert, K. Willimann, A. Blaser, R. S. Roos, P. Loetscher, and B. Moser A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells J. Exp. Med., May 3, 2004; 199(9): 1265 - 1275. [Abstract] [Full Text] [PDF]

				A. Cavani, F. Nasorri, C. Ottaviani, S. Sebastiani, O. De Pita, and G. Girolomoni Human CD25+ Regulatory T Cells Maintain Immune Tolerance to Nickel in Healthy, Nonallergic Individuals J. Immunol., December 1, 2003; 171(11): 5760 - 5768. [Abstract] [Full Text] [PDF]

				S. Mitola, M. Strasly, M. Prato, P. Ghia, and F. Bussolino IL-12 Regulates an Endothelial Cell-Lymphocyte Network: Effect on Metalloproteinase-9 Production J. Immunol., October 1, 2003; 171(7): 3725 - 3733. [Abstract] [Full Text] [PDF]

				J. Kao, J. Kobashigawa, M. C. Fishbein, W. R. MacLellan, M. D. Burdick, J. A. Belperio, and R. M. Strieter Elevated Serum Levels of the CXCR3 Chemokine ITAC Are Associated With the Development of Transplant Coronary Artery Disease Circulation, April 22, 2003; 107(15): 1958 - 1961. [Abstract] [Full Text] [PDF]

				A. Y. Savinov, F. S. Wong, A. C. Stonebraker, and A. V. Chervonsky Presentation of Antigen by Endothelial Cells and Chemoattraction Are Required for Homing of Insulin-specific CD8+ T Cells J. Exp. Med., March 3, 2003; 197(5): 643 - 656. [Abstract] [Full Text] [PDF]

				R. Mo, J. Chen, Y. Han, C. Bueno-Cannizares, D. E. Misek, P. A. Lescure, S. Hanash, and R. L. Yung T Cell Chemokine Receptor Expression in Aging J. Immunol., January 15, 2003; 170(2): 895 - 904. [Abstract] [Full Text] [PDF]

				C. D. Chung, F. Kuo, J. Kumer, A. S. Motani, C. E. Lawrence, W. R. Henderson Jr., and C. Venkataraman CCR8 Is Not Essential for the Development of Inflammation in a Mouse Model of Allergic Airway Disease J. Immunol., January 1, 2003; 170(1): 581 - 587. [Abstract] [Full Text] [PDF]

				G. Penna, M. Vulcano, A. Roncari, F. Facchetti, S. Sozzani, and L. Adorini Cutting Edge: Differential Chemokine Production by Myeloid and Plasmacytoid Dendritic Cells J. Immunol., December 15, 2002; 169(12): 6673 - 6676. [Abstract] [Full Text] [PDF]

				T. Nakajima, N. Inagaki, H. Tanaka, A. Tanaka, M. Yoshikawa, M. Tamari, K. Hasegawa, K. Matsumoto, H. Tachimoto, M. Ebisawa, et al. Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcepsilon receptor I cross-linking: an interspecies comparison Blood, December 1, 2002; 100(12): 3861 - 3868. [Abstract] [Full Text] [PDF]

				R. Stephens, D. A. Randolph, G. Huang, M. J. Holtzman, and D. D. Chaplin Antigen-Nonspecific Recruitment of Th2 Cells to the Lung as a Mechanism for Viral Infection-Induced Allergic Asthma J. Immunol., November 15, 2002; 169(10): 5458 - 5467. [Abstract] [Full Text] [PDF]

				R. Stephens and D. D. Chaplin IgE Cross-Linking or Lipopolysaccharide Treatment Induces Recruitment of Th2 Cells to the Lung in the Absence of Specific Antigen J. Immunol., November 15, 2002; 169(10): 5468 - 5476. [Abstract] [Full Text] [PDF]

				H. Akiba, J. Kehren, M.-T. Ducluzeau, M. Krasteva, F. Horand, D. Kaiserlian, F. Kaneko, and J.-F. Nicolas Skin Inflammation During Contact Hypersensitivity Is Mediated by Early Recruitment of CD8+ T Cytotoxic 1 Cells Inducing Keratinocyte Apoptosis J. Immunol., March 15, 2002; 168(6): 3079 - 3087. [Abstract] [Full Text] [PDF]

				H. K. Maxion and K. A. Kelly Chemokine Expression Patterns Differ within Anatomically Distinct Regions of the Genital Tract during Chlamydia trachomatis Infection Infect. Immun., March 1, 2002; 70(3): 1538 - 1546. [Abstract] [Full Text] [PDF]

				P. McGuirk, C. McCann, and K. H.G. Mills Pathogen-specific T Regulatory 1 Cells Induced in the Respiratory Tract by a Bacterial Molecule that Stimulates Interleukin 10 Production by Dendritic Cells: A Novel Strategy for Evasion of Protective T Helper Type 1 Responses by Bordetella pertussis J. Exp. Med., January 22, 2002; 195(2): 221 - 231. [Abstract] [Full Text] [PDF]

				S. Artik, K. Haarhuis, X. Wu, J. Begerow, and E. Gleichmann Tolerance to Nickel: Oral Nickel Administration Induces a High Frequency of Anergic T Cells with Persistent Suppressor Activity J. Immunol., December 15, 2001; 167(12): 6794 - 6803. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sebastiani, S. Articles by Cavani, A. Search for Related Content PubMed PubMed Citation Articles by Sebastiani, S. Articles by Cavani, A.