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International Immunology Advance Access published online on August 13, 2007
International Immunology, doi:10.1093/intimm/dxm066
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Dose dependence of oral tolerance to nickel

Xianzhu Wu1,3, Karin Roelofs-Haarhuis1, Jianhong Zhang1, Michael Nowak1,4, Laura Layland1,2, Erich Jermann1 and Ernst Gleichmann1

1 Institut für Umweltmedizinische Forschung at Heinrich Heine University gGmbH, Düsseldorf, Auf'm Hennekamp 50, D-40225 Düsseldorf, Germany
2 Institute for Medical Microbiology, Immunology and Hygiene at Technical University of Munich, Germany
3 Present address: Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
4 Present address: Beth Israel Deaconess Medical Center, Hematology/Oncology division, Harvard Medical School, Boston MA, 02215, USA

Correspondence to: Correspondence to: E. Gleichmann; E-mail: ernst.gleichmann{at}uni-duesseldorf.de


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
The dose dependence of oral nickel tolerance was analyzed by comparing three different subsets of C57BL/6 mice: Nivery low mice were reared in a nickel-reduced environment, Nilow and Nihigh mice were reared in a stainless steel-containing environment and the latter received oral NiCl2 (10 mM). In spleen and feces, Nivery low mice exhibit significantly lower nickel concentrations than Nilow and Nihigh mice. In contrast to Nivery low mice that can be sensitized with a single intradermal administration of NiCl2 alone, Nilow mice can only be sensitized in the presence of an adjuvant and Nihigh mice cannot be sensitized at all. This dose-dependent resistance to nickel sensitization (i.e. Nihigh > Nilow > Nivery low) correlates with differences in the number and type of nickel-specific T regulatory (Treg) cells. Adoptive transfer studies into Nivery low recipients showed that Nivery low mice completely lack specific Treg cells whereas Nilow and Nihigh mice harbor them, albeit their numbers and/or suppressive strength are much higher in Nihigh than Nilow mice. The principal Treg subset in Nilow mice consists of CD4+CD25+ cells, among which CD4+CD25+{alpha}Eß7+ cells are the most effective. In Nihigh mice, CD4+CD25+ Treg cells co-exist with an ensemble of CD8+ Treg and CD4+CD25– suppressor–inducer cells.

Keywords: allergy, nickel, regulatory T cells, tolerance/suppression


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Nickel is one of the most frequent contact allergens. It is a constituent of various different alloys and, hence, occurs in numerous metal items used in life, from which it can be released in varying amounts in the presence of bodily fluids, including sweat and saliva (1). Nickel ions can generate neoantigens, or signal 1, needed for the activation of nickel-specific T cells. Whether the obligatory co-stimulation (signal 2) required for T cell priming is provided by an adjuvant effect of nickel ions themselves or by a variety of different cofactors subsumed under the term ‘danger’ (2) remains an unresolved issue. The latter possibility is supported by the clinical experience that nickel allergy preferentially develops after nickel has been in contact with an inflamed or irritated skin, whereas sensitization is unlikely to occur with the intact skin. Consistent with this, the majority of humans fails to become sensitized to nickel despite its ubiquitous occurrence (3), and interestingly, they possess nickel-specific CD4+CD25+ T regulatory (Treg) cells (4). In fact, even though a reduction in dermal nickel exposure has been shown to decrease the incidence of newly sensitized nickel allergics (3), other epidemiological observations suggest that nickel hypersensitivity is lowered in the presence of elevated environmental levels of this potential sensitizer (5).

In both humans and experimental animals, most of the bodily absorption of nickel is through oral consumption (6), a route known to obviate the induction of contact hypersensitivity (CHS) for some other allergens and to favor the induction of Treg cells and tolerance (7). Consistent with this, an increased oral nickel uptake rendered experimental animals unresponsive to the induction of nickel allergy (8–10). Moreover, there is solid epidemiological evidence to indicate that in at least some wearers of dental braces, nickel tolerance may be acquired due to the continuous release of low amounts of nickel from such braces (11). Interestingly, however, the amount of nickel released from the braces is negligible when compared with the total daily uptake from food (~0.3 ng–1kg–1day–1 versus 2–4 µg–1kg–1day–1) (1, 12, 13). This paradox raises questions about the potential effects of different exposure conditions and the dose–response relationship of oral nickel tolerance and nickel-specific Treg cells, respectively. Treg cells specific for nickel have been described as CD4+CD25+ in non-allergic humans (4). In mice exposed to a high concentration of oral nickel, both CD4+ and CD8+ T cells are required for the protection from sensitization with NiCl2 combined with the adjuvant hydrogen peroxide (H2O2) (8), whether or not CD4+CD25+ Treg cells also are involved has not been studied so far.

To further clarify the conditions that allow the development of nickel hypersensitivity or nickel tolerance, we have performed a systematic analysis of the dose dependence required for oral nickel tolerance by comparing three types of C57BL/6 mice which were termed Nihigh, Nilow and Nivery low due to their different oral exposure to nickel. Our results indicate that there is a hierarchy Nivery low > Nilow > Nihigh mice with respect to the ease of sensitization to nickel and a reverse hierarchy Nihigh > Nilow > Nivery low with respect to nickel-specific Treg cells. Moreover, we analyzed to which extent oral nickel tolerance is mediated by CD4+CD25+ and CD8+ Treg cells. Whereas CD4+CD25+ Treg cells proved capable of preventing sensitization induced by NiCl2 alone (i.e. without additional adjuvant), an ensemble of CD8+ Treg cells and CD4+CD25– T cells was needed to prevent the hypersensitivity resulting from the vigorous immunization with NiCl2/H2O2.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Reagents
NiCl2·6H2O (denoted as NiCl2) and 2,4-dinitrofluorobenzene (DNFB) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany), and H2O2 was obtained from E. Merck (Darmstadt, Germany).

Antibodies
Allophycocyanin (APC)-labeled streptavidin and the following anti-mouse antibodies were purchased from BD PharMingen (Heidelberg, Germany): APC–anti-CD3{epsilon} (145-2C11), PerCP–anti-CD4 (RM4-5), PE– and biotin–anti-CD8ß.2 (53-5.8), purified anti-CD16/CD32 (2.4G2), FITC–anti-CD25 (7D4), PE–anti-CD25 (PC61), PE–anti-{alpha}IEL chain (M290), PE–anti-I-A/I-E (M5/114.15.2) and FITC–anti-TCRß chain (H57-597). Anti-CD25 mAb was harvested from hybridoma PC61 (14). The magnetically labeled anti-mouse MHC-II, anti-mouse CD4 and anti-PE microbeads were obtained from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).

Mice
Nilow mice were specific pathogen-free C57BL/6J mice obtained from Janvier (Le Genest St Isle, France). They were bred and reared in a conventional environment with cage covers and water bottle tips made from nickel-containing stainless steel (grade 304; DIN no. 14301); they received tap water without additional nickel supplementation. Nivery low mice were generated by breeding and rearing Nilow mice for two generations in a nickel-reduced environment using cage covers and water bottle lids made of plastic and glass, respectively. Nihigh mice were generated by giving Nilow mice 10 mM NiCl2 in the drinking water for 4–6 weeks.

All mice received a standard rodent diet (Sniff Spezialdiaeten, Soest, Germany) and were kept in accordance with the animal husbandry, as described in previous papers (8, 15). They were 7–10 weeks old at the onset of experiments. All investigations were carried out using female mice.

Determination of nickel concentration
Nickel concentration in urine, feces and organs of individual animals were determined by atomic absorption spectroscopy using a Zeeman 4100 ZL spectrometer (PerkinElmer, Boston, MA, USA). In the case of Nihigh mice, the excretions and bodily organs were obtained 1 day after cessation of a 4-week period of oral nickel administration, and age-matched Nilow and Nivery low mice were used as controls. The animals (three to five mice per group) were bled by heart puncture, and the organs and feces were handled with teflon-coated instruments to avoid contamination by nickel ions released from stainless steel instruments. To further reduce contamination by nickel ions, plastic pipettes and sample collection tubes were washed with 10% NH4Cl, rinsed with the same aqua bidest as that used for atomic absorption spectroscopy and dried in a heating oven overnight. Samples were prepared for the analysis by high-pressure ashing.

Sensitization of mice
Mice were primed as described previously (8) with minor modifications. In the case of nickel, Nilow mice were injected intradermally (i.d.) into both flanks (50 µl each) with either sterile, pyrogen-free saline, 10 mM NiCl2 in saline or 10 mM NiCl2 in saline containing 1% H2O2. When priming Nivery low mice, however, 50 µl of either 1 mM NiCl2 in saline or saline containing 1% H2O2 was used. In the case of DNFB, mice were primed by painting 0.5% (w/v) DNFB onto shaved flanks (25 µl each); DNFB was solved in a 4:1 (v/v) mixture of acetone and olive oil, respectively.

Challenge for recall and ear-swelling test
Ten days after priming, mice were challenged for recall by injecting 50 µl of 10 mM NiCl2 in sterile, pyrogen-free saline into the pinna of each ear or by applying 50 µl of 0.2% DNFB onto each ear. Forty-eight hours after challenge with NiCl2 and 24 h after challenge with DNFB, delayed-type hypersensitivity (DTH) reactions were determined by measuring the increment in ear thickness compared with pre-challenge values. For determination of pre-challenge values, mice were anesthetized; for measurement post-challenge, they were sacrificed by asphyxiation with CO2. Measurements were performed in a blind fashion using a micrometer (Oditest D 1000 gauge, The Dyer Co., Lancaster, PA, USA). The data shown represent individual experiments comprising groups of five to six mice each, the mean ear-swelling response is expressed in units of mm x 10–2 + SEM.

Adoptive T cell transfer
In all transfer experiments, Nivery low mice (five to six animals per group) were used as recipients. Suspensions of the indicated type and number cell were injected intravenously (i.v.) into recipient mice (150 µl per mouse). To study the ability of transferred T cells in preventing subsequent sensitization to nickel, recipients were primed i.d. 1 day after adoptive transfer; 10 days after priming, they were challenged for recall at the ears, and 48 h after recall, their ear-swelling response was measured. This experimental approach was used for all experiments except that shown in Fig. 4B in which the capacity of transferred T cells to suppress the elicitation of hypersensitivity in nickel-sensitized mice was studied. Here, recipients were primed 10 days prior to transfer; 1 day after transfer, they were challenged at the ears and 48 h later ear-swelling responses were determined.


Figure 4
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  		Fig. 4. The minimal number of T cells required for preventing post-transfer sensitization of Nivery low recipients is determined by both the type of T cell donor and the mode of recipient immunization. Indicated numbers of unsorted T cells from Nihigh (A and B) and Nilow (C) donor mice were transferred and the recipients primed and challenged, as indicated. Saline-injected Nivery low mice served as negative control groups throughout. (A) Demonstrates the number of Nihigh donor T cells required to prevent sensitization after priming with NiCl2 alone. (B) Shows the number of Nihigh donor T cells required to prevent sensitization after priming with NiCl2/H2O2. (C) Depicts the number of Nilow donor T cells required to prevent sensitization after priming with NiCl2 alone. Data represent one of two independent experiments showing comparable results.

 
Cell enrichment and sorting
To enrich donor T cells, single-cell suspensions of erythrocyte-free splenocytes were prepared in complete RPMI medium (PAA Laboratories, Coelbe, Germany) containing 10% FCS and passed through nylon wool columns. Eluted T cells were then further purified by either depletion of MHC-II+ cells to obtain total T cells or depletion of MHC-II+ cells followed by positive selection of CD4+ or CD8+ T cells using MACS microbeads labeled with the respective mAbs (Miltenyi Biotec GmbH). The sorted T cells were contaminated with <1% MHC-II+ cells; the sorted CD4+ and CD8+ T cells contained <1% CD8+ and <2% CD4+ T cells, respectively. In some experiments, the sorted CD8+ and CD4+ T cells were recombined in a 1:1 ratio prior to injection into the recipients.

To obtain CD4+{alpha}Eß7– T cells, sorted CD4+ T cells were incubated with PE-labeled mAb against {alpha}E integrin (CD103), followed by anti-PE microbeads. After depletion of {alpha}Eß7+ T cells, the remaining contamination by such cells in the CD4+CD25+ T cell subset was ~4%.

Flow cytometry
For phenotyping T cells, single-cell suspensions were prepared in PBS and pre-incubated with anti-CD16/CD32 mAb (Fc block), before staining with the respective antibodies. For the determination of total CD4+CD25+ T cells (Fig. 8A) and the population of {alpha}Eß7+ T cells among CD4+CD25+ T cells (Fig. 8B), 15 and 13 mice were individually analyzed. Flow cytometry was performed using a FACSCalibur (BD Biosciences, Heidelberg, Germany), and the results were analyzed with CellQuest software.


Figure 8
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  		Fig. 8. Increasing oral nickel uptake augments both the percentage of CD25+cells within the CD4+T cell population and the percentage of {alpha}Eß7+T cells among CD4+CD25+T cells. Splenic CD4+and CD4+CD25+T cells from the indicated mouse subsets were gated, and the percentages of CD25+cells within CD4+T cells (left panel) and {alpha}Eß7+T cells among CD4+CD25+T cells (right panel) were determined.

 
In vivo depletion of CD25+ cells
Hybridoma PC61 was grown to over-confluent levels in DMEM medium (PAA Laboratories) supplemented with 10% ultra-low IgG FCS (PAN Biotech GmbH, Aidenbach, Germany). Anti-CD25 mAb was then obtained using a Hitrap Protein G column with an ÄKTA prime (Amersham Pharmacia Biotech, Uppsala, Sweden). After concentrating the antibody molecules using centripreps (Millipore GmbH, Eschborn, Germany), the concentration levels were determined by standard protein assays. Purified anti-CD25 mAb (0.25 mg per mouse) was injected intra-peritoneally (i.p.) into either Nihigh or Nilow mice, and 3 days later the mice were sacrificed and their spleen cells isolated, as described above.

Statistical analysis
Statistical significance of results was determined by analysis of variance (ANOVA) followed by Newman–Keuls test. One-way ANOVA tests were performed using GraphPad Prism (GraphPad software, San Diego, CA, USA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Analysis of nickel concentration in tissues and excretions from Nivery low, Nilow and Nihigh mice
Concentrations of nickel found in the tissues and excretions from Nihigh mice obtained directly after oral NiCl2 treatment were stopped far exceeded those found in Nilow and Nivery low mice. When comparing the latter two mouse subsets, statistically significant differences were only found in the feces and spleen. In both cases, the values measured in Nilow mice exceeded those in the Nivery low mice.

Unlike Nilow mice, Nivery low mice can be sensitized by NiCl2 alone
Differences in the susceptibility of Nihigh and Nilow mice to become sensitized toward nickel have already been reported (8, 16, 17). Here, we asked whether or not there is a corresponding difference between Nilow and Nivery low mice. To increase skin inflammation and irritation, we used intradermal rather than epicutaneous application to induce hypersensitivity to nickel (15). In confirmation with earlier results (8, 16, 17), a re-challenge in the ears with NiCl2 via intradermal application resulted in only background ear swelling in Nilow mice primed with NiCl2 alone, but elicited significantly higher responses in Nilow mice sensitized with NiCl2/H2O2 (Fig. 2, bar 1 versus bar 2). In marked contrast, the Nivery low mice could be sensitized with NiCl2/H2O2 (Fig. 2, bar 7) and NiCl2 alone (Fig. 2, bar 6). In either case, the immune response of Nivery low mice was nickel specific, because these nickel-primed mice failed to elicit an ear-swelling response when recalled with DNFB (Fig. 2, bars 9 and 10). Moreover, DNFB-immunized mice showed high ear-swelling responses when recalled with DNFB (Fig. 2, bar 11), but not NiCl2 (Fig. 2, bar 8).


Figure 2
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  		Fig. 2. Unlike Nilow mice, Nivery low mice can be sensitized by exposure to nickel ions alone (i.e. without the need for co-administration of the adjuvant H2O2). Nilow and Nivery low mice were primed with saline, NiCl2 alone or NiCl2 in H2O2, as indicated, and control groups were painted with DNFB. They were challenged for recall, as indicated, and their ear-swelling responses determined. The data shown represent one of three independent experiments showing comparable results. In this and the following figures, any reference to bar numbers in the text refers to the order of the bars read from top to bottom. Additionally, black bars indicate the critical experimental groups.

 
Nilow T cells suppress both the induction and elicitation of nickel hypersensitivity in Nivery low recipients immunized with NiCl2 alone
Nihigh mice proved resistant to sensitization not only with NiCl2 alone but also with NiCl2/H2O2; moreover, their T cells were anergic upon re-stimulation with NiCl2 in vitro and able to transfer nickel tolerance to Nilow recipients. In contrast, Nilow mice were susceptible to sensitization with NiCl2/H2O2, but not to sensitization with NiCl2 alone (8, 15). Thus, we asked whether Nilow mice possessed nickel-specific Treg cells which provided this partial resistance to nickel sensitization. To test this, T cells isolated from Nihigh, Nilow or Nivery low donors were transferred into naive Nivery low recipients which were subsequently immunized, challenged and tested (Fig. 3A). Indeed, Nilow donors provided nickel-specific Treg cells to the Nivery low recipients that protected the latter from being sensitized by NiCl2 alone (Fig. 3A, bar 5 versus bar 1). Interestingly, these Nilow T cells failed to protect Nivery low recipients that were immunized with NiCl2/H2O2 (Fig. 3A, bar 6), indicating that the latter way of immunization against nickel is more difficult to suppress than the former. In contrast, transferred Nivery low T cells completely failed to suppress the induction of nickel-induced hypersensitivity (Fig. 3A, bars 1 and 2). As expected, Nihigh T cells not only prevented the sensitization induced with NiCl2 alone (shown in Fig. 6E, bar 3) but also that induced with NiCl2/H2O2 (Fig. 3A, bar 3). The suppression exerted by the transferred Nilow T cells was nickel specific (Fig. 3A, bar 7 versus 9); the specificity of Treg cells from Nihigh mice has been demonstrated elsewhere (8).


Figure 3
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  		Fig. 3. Adoptively transferred Nilow T cells suppress the nickel hypersensitivity induced by immunization with NiCl2 alone, whereas the suppressive capacity of Nihigh T cells is not confined by the mode of recipient immunization. Splenic T cells (107 per recipient) from Nihigh, Nilow or Nivery low donors were transferred into Nivery low recipients. (A) Showing the effect of donor T cells on recipients that were primed and challenged for recall after the cell transfer. (B) Showing the effect of donor T cells on recipients that were primed before the transfer and challenged for recall thereafter. Data represent one of four (A) or two (B) independent experiments showing comparable results.

 

Figure 6
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  		Fig. 6. Whereas CD4+CD25+ T cells from nickel-exposed donors are able to suppress NiCl2 alone-induced sensitization, both CD4+CD25– and CD8+ T cells are required to prevent sensitization by NiCl2/H2O2. (A) Indicating the percentage of CD4+CD25+ T cells in the spleen of untreated Nihigh mice (left panel) and Nihigh mice that had received an i.p. injection of anti-CD25 mAb (right panel). (B–E) Nilow and Nihigh were injected with anti-CD25 mAb or left untreated, as indicated. Specified numbers and T cell subsets from the indicated donor mice were then transferred into Nivery low recipients. Equivalent numbers and cell types obtained from Nivery low donor mice served as non-suppressive control cells. After recipients were primed, as indicated, and challenged with NiCl2, their ear-swelling response was determined. Data represent one of two independent experiments showing comparable results.

 
Knowing that Treg cells from Nihigh and Nilow mice can completely or partially prevent the post-transfer sensitization toward nickel, respectively (Fig. 3A), we asked whether they were also able to inhibit the elicitation of nickel hypersensitivity in already sensitized mice. Therefore, T cells from Nihigh, Nilow or Nivery low donors were transferred into Nivery low recipients that were sensitized 10 days before transfer (Fig. 3B). In contrast to non-suppressive Nivery low T cells, Nihigh T cells profoundly suppressed the elicitation of nickel hypersensitivity and this was irrespective of how hypersensitivity was induced (Fig. 3B, bars 4 and 5). Nilow T cells were equally able to suppress nickel hypersensitivity in Nivery low recipients sensitized with NiCl2 alone (Fig. 3B, bar 7). In recipients immunized with NiCl2/H2O2, donor Nilow T cells demonstrated an intermediate suppressive capacity (Fig. 3B, bar 8). From the results shown in Fig. 3, we can conclude that the increase in oral nickel uptake correlates with the suppressive capacity of the transferred T cells.

Origin of T cell and sensitization determine the minimal number of T cells required for transferring nickel unresponsiveness to Nivery low mice
In Fig. 3, Nivery low recipients received 107 enriched donor T cells. However, previous studies using Nilow recipients demonstrated that tolerance could be transferred with only 102 Nihigh donor T cells (8). Consequently, we wondered how few donor T cells were actually required for the transfer of unresponsiveness to Nivery low recipients. To establish this minimal number, we performed dose–response experiments using Nihigh and Nilow donor T cells. Following immunization with NiCl2 alone, it became apparent that 103 Nihigh T cells were sufficient to tolerize Nivery low recipients (Fig. 4A, bar 4). Interestingly, 104 T cells from the same donors were required when recipients were immunized with NiCl2/H2O2 (Fig. 4B, bar 5). Furthermore, whereas 103 Nihigh T cells sufficed to transfer the tolerance to Nivery low recipients subsequently immunized with NiCl2 alone (Fig. 4A, bar 4), as many as 106 Nilow T cells were required to achieve the same effect (Fig. 4C, bar 7). Clearly, the number and/or type of nickel-specific Treg cells generated within Nihigh mice are superior to those from Nilow mice.

Differential suppressive capacity of CD4+ and CD8+ T cells from Nihigh and Nilow donors
Previously, we reported that both CD4+ and CD8+ Nihigh donor T cells were required for a successful transfer of tolerance into Nilow recipients that were subsequently immunized with NiCl2/H2O2 (8) (Fig. 5A, bar 8). We have now used the same experimental approach in order to compare the effects of CD4+ and CD8+ T cell sub-populations obtained from Nilow donors. As anticipated (cf. Fig. 3), T cell subsets from Nivery low donors, or combinations thereof, were unable to prevent the sensitization at any of the cell numbers transferred (Fig. 5A–C). In contrast to CD8+ T cells, CD4+ T cells from Nihigh (103) and Nilow (106) donors were able to prevent post-transfer sensitization by NiCl2 alone (bar 4 in Fig. 5B and C, respectively). In contrast, when the recipients were immunized with NiCl2/H2O2, a transfer of even 104 Nihigh CD4+ T cells still failed to prevent their sensitization (Fig. 5A, bar 4). In fact, CD8+ T cells from these donors were devoid of any suppressive capacity when transferred alone (bar 6 in Fig. 5A–C). These results confirmed our previous findings that the sensitization induced by NiCl2 alone can be more readily suppressed than that induced by NiCl2/H2O2 (Figs 3–5) and that Nihigh CD4+ T cells alone are unable to prevent sensitization with NiCl2/H2O2 (8).


Figure 5
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  		Fig. 5. Both Nihigh and Nilow CD4+ T cells prevent nickel hypersensitivity induced by NiCl2 alone, but combined CD4+ and CD8+ T cells from Nihigh donors are needed to prevent the hypersensitivity induced by NiCl2/H2O2. (A–C) T cells from Nivery low, Nilow or Nihigh donor mice were transferred, as indicated. They were transferred as unsorted cells (bars 1 and 2), sorted CD4+ T cells (bars 3 and 4), sorted CD8+ cells (bars 5 and 6) or as an ensemble of sorted CD4+ and CD8+ T cells that were recombined at a 1:1 ratio prior to injection to yield the number of transferred T cells indicated (bars 7 and 8). In (A) 104 T cells were transferred and recipients primed with NiCl2/H2O2 after transfer, while in (B) 103 and in (C) 106 T cells were transferred and recipients primed with NiCl2 alone. All recipients were challenged with NiCl2, and their ear-swelling response was determined. Data represent one of two independent experiments showing comparable results.

 
Although we have previously shown that 102 antigen-presenting cells from Nihigh donor mice are sufficient in transferring tolerance to Nilow recipients (8), this contaminating population is not responsible for the prevention of hypersensitivity in Nivery low recipients upon transfer of 104 CD4+/CD8+ T cells from Nihigh mice (Fig. 5A, bar 8). This conclusion is drawn from the results obtained from the transfer of the individual T cell subsets (104) which were unable to prevent hypersensitivity (Fig. 5A, bars 4 and 8).

Taken together, the data shown in Fig. 5 demonstrate that nickel tolerance, as induced by adoptive cell transfer into Nivery low recipients, is a fine blend between the mode of recipient immunization (NiCl2 alone or with H2O2), the amount of oral nickel exposure of the T cell donor and the transferred T cell subsets (CD8+ and/or CD4+).

Suppressive activity of CD4+CD25+ Treg cells
Next, we studied whether the protection from transferred donor CD4+ T cells (bar 4 of Fig. 5B and C) was provided by the CD4+CD25+ population (18). Thus, Nihigh (Fig. 6A) and Nilow (data not shown) donors were depleted of CD25+ T cells by injection of anti-CD25 mAb. Based on the information obtained earlier, we transferred either 103 CD4+ Nihigh T cells (Fig. 6B), 104 unsorted Nihigh T cells (Fig. 6C), 103 unsorted Nihigh T cells (Fig. 6E) or 106 CD4+ Nilow T cells (Fig. 6D); the Nivery low recipients were immunized after transfer with either NiCl2/H2O2 (Fig. 6C) or NiCl2 alone (Fig. 6B, D and E). Indeed, the cell types and numbers specified sufficed to transfer nickel tolerance when the donor mice had not been depleted of CD25+ cells prior to transfer (bar 3 of Fig. 6B–E). However, when the CD4+ T cells from either Nihigh or Nilow donors were depleted of CD25+ cells, their capacity to prevent the sensitization by NiCl2 alone was alleviated (bar 2 in Fig. 6B and D). Therefore, Nihigh and Nilow mice possess CD4+CD25+ Treg cells which are able to prevent nickel hypersensitivity induced by NiCl2 in Nivery low recipients.

A different picture emerged when unsorted Nihigh T cells, instead of CD4+ Nihigh T cells, were depleted of CD25+ T cells and then transferred. In this case, the depletion of CD25+ cells failed to alleviate the suppressive capacity and this was regardless of whether the recipients were immunized with NiCl2/H2O2 or NiCl2 alone (bar 2 in Fig. 6C and E). It should be realized here that the unsorted Nihigh T cells depleted of CD25+ cells contained CD4+CD25– and CD8+ T cells. As shown above, the nickel hypersensitivity in recipients immunized with either NiCl2/H2O2 or NiCl2 alone could not be prevented by the sole transfer of Nihigh CD8+ T cells (bar 6 of Fig. 5A and B), but required the co-transfer of Nihigh CD4+ T cells (Fig. 5A, bar 8). Therefore, it is conceivable that the maintenance of suppressor activity by unsorted CD25– Nihigh T cells (bar 2 in Fig. 6C and E) was due to the preservation within that population of CD8+ Treg cells and that these cells received help from CD4+CD25– T cells acting as so-called T suppressor–inducer cells.

Suppressive activity of CD4+CD25+{alpha}Eß7+ Treg cells
CD4+CD25+ T cells expressing the integrin {alpha}E chain, a component of the homing receptor {alpha}Eß7+ integrin or CD103, were reported to have a somewhat stronger suppressive capacity than CD4+CD25+{alpha}Eß7– or CD4+CD25–{alpha}Eß7+ T cells (19). To determine whether CD4+CD25+{alpha}Eß7+ T cells were the main population that can suppress nickel hypersensitivity, CD4+ or CD4+{alpha}Eß7– T cells from Nihigh or Nilow donor mice were transferred into Nivery low recipients. Confirming the results described above (Figs 5 and 6), CD4+ T cells from both Nilow and Nihigh donors completely suppressed nickel hypersensitivity induced by NiCl2 alone (Fig. 7, bars 2 and 4). When {alpha}Eß7+ cells from these CD4+ T cells were depleted, however, the suppression partially disappeared (Fig. 7, bars 3 and 5). Together with the results shown in Fig. 6, it becomes apparent that the suppression of nickel hypersensitivity induced by NiCl2 alone was provided by CD4+CD25+ T cells from Nihigh and Nilow mice and that within that population the CD4+CD25+{alpha}Eß7+ T cells have the most suppressive capacity.


Figure 7
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  		Fig. 7. CD4+CD25+{alpha}Eß7+ T cells from Nihigh and Nilow donors can prevent the sensitization induced by NiCl2 alone. Indicated numbers and subsets of T cells were obtained from Nivery low, Nilow or Nihigh donors and transferred as either sorted CD4+ cells (bars 1, 2 and 4) or sorted CD4+{alpha}Eß7– cells (bars 3 and 5). After the recipients were primed and challenged with NiCl2, their ear-swelling response was determined. Data represent one of three independent experiments showing comparable results.

 
Oral nickel uptake dose dependently enhances the percentage of splenic CD4+CD25+{alpha}Eß7+ T cells
As far as immunization with NiCl2 alone is concerned, the data presented here indicate that a 1000 times more Nilow than Nihigh CD4+ donor T cells are required to prevent sensitization (Fig. 4A and C). In addition, the major sub-populations involved are CD4+CD25+ and CD4+CD25+{alpha}Eß7+ T cells (Figs 6B, D and 7), two phenotypes associated with Treg cells. Thus, we asked whether the differential suppressive capacity of Nihigh and Nilow CD4+ T cells could be due to a higher frequency of these Treg sub-populations in Nihigh than Nilow mice. In order to test this, we analyzed the spleens of uninjected Nihigh, Nilow and Nivery low mice for the frequencies of CD4+CD25+ and CD4+CD25+{alpha}Eß7+ T cells. As shown in Fig. 8 (left panel), an increased oral uptake of nickel (cf. Fig. 1) resulted in an increased percentage of splenic CD4+CD25+ T cells. Similarly, there were also significant increases with nickel intake in the percentage of {alpha}Eß7+ cells among the CD4+CD25+ T cells of Nihigh, Nilow and Nivery low mice (Fig. 8, right panel). Together, the results in Figs 7 and 8 suggest that the stronger suppressive capacity of Nihigh CD4+ T cells was due to the higher frequency of CD4+CD25+{alpha}Eß7+ Treg cells observed in these animals.


Figure 1
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  		Fig. 1. Nickel concentrations in excretions and organs of Nivery low, Nilow and Nihigh mice. Results represent one of two experiments yielding very similar results. In this and the following figures, asterisks denote a significant statistical difference (*P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
With our three types of nickel-exposed C57BL/6 mice, we have established that there is a clear-cut inverse dose–response relationship between the amount of oral nickel uptake and the ease in which mice can be sensitized to nickel. To summarize, whereas Nihigh mice could not be sensitized to nickel at all, Nivery low mice could be sensitized even with NiCl2 alone and Nilow mice with NiCl2/H2O2. This hierarchy of Nivery low > Nilow > Nihigh mice with respect to the ease of sensitization was mirror imaged by the hierarchy of Nihigh > Nilow > Nivery low with regard to the number and the suppressive capacity of nickel-specific Treg cells detected in these mice.

Since Nilow mice received the same food and drinking water as Nivery low mice, the only additional nickel ions accessible to the former were those released from their stainless steel cage covers (by saliva and gnawing) and drinking bottle lids (through water contact and licking). This elevated oral nickel uptake of Nilow mice was verified by their small but significantly higher nickel concentration in the spleen and feces. This background level of nickel is enough to induce a state of partial tolerance which protects Nilow mice from being sensitized by nickel ions alone, albeit not by nickel ions combined with the adjuvant H2O2 (or other adjuvants) (15; Fig. 2). A similar scenario exists in humans: continuous exposure to nickel in the oral cavity (from orthodontic braces) was found to correlate with a decreased incidence of nickel allergy (20). A possible explanation for this partial nickel tolerance of Nilow mice and wearers of orthodontic braces could be that, as in hamsters (21), the mucosa of the cheek pouch of mice and men is an immunologically privileged site.

The use of Nivery low mice as recipients of adoptively transferred T cells enabled us to compare the suppressive capacity of nickel-specific T cells obtained from either Nihigh or Nilow donors. Due to the following two criteria, we conclude that unsorted T cells from Nihigh donors contain more potent nickel-specific Treg cells than those from Nilow mice. First, the required number of unsorted Nihigh T cells to prevent induction of nickel hypersensitivity is much lower than that of Nilow donors. Second, the capacity of unsorted Nihigh Treg cells to prevent the subsequent induction of nickel hypersensitivity and to preclude its elicitation in pre-sensitized recipients functioned independently from the type of immunization (i.e. NiCl2 alone or with H2O2) of the latter. In contrast, the suppressive capacity of Nilow Treg cells depended on the mode of recipient sensitization. Unlike Nihigh and Nilow mice, the Nivery low mice were found to completely lack nickel-specific Treg cells. Interestingly, regulatory T cells from Nihigh donor mice were able to suppress both primary and secondary responses to nickel (Fig. 3A, bar 3 and Fig. B, bar 5). In contrast, Nilow Treg cells that were unable to suppress the subsequent immunization with NiCl2/H2O2 (Fig. 3A, bar 6) were also unable to inhibit a secondary response after sensitization with NiCl2/H2O2 (Fig. 3B, bar 7). Collectively, these data support the notion that nickel-specific regulatory T cells are not only able to suppress the immunization within a time frame of 10 days but also secondary immune responses within 2 days upon adoptive transfer.

The suppressive capacity of CD4+CD25+ Treg cells has been widely established (18). We demonstrate here that while donor Treg cells from Nihigh or Nilow mice could prevent hypersensitivity induced by NiCl2 alone, CD4+CD25– T cells failed to do so. However, the CD4+CD25+ Treg cell-containing CD4+ T cells failed to prevent the hypersensitivity inducible by NiCl2/H2O2. Thus, vigorous immunization can break the incomplete tolerant state provided by CD4+CD25+ Treg cells. This conclusion correlates to findings made in vitro showing that the suppressive activity of CD4+CD25+ Treg cells can be overruled by providing a more potent signal 2 (22, 23).

Although CD4+ T cells (containing CD4+CD25+ Treg cells) from both Nihigh and Nilow mice prevented nickel hypersensitivity induced by NiCl2 alone, the number of effective CD4+ T cells required from Nilow mice was 1000 times higher than that needed from Nihigh donors. Interestingly, when analyzing the percentage of CD25+ cells among CD4+ T cells and the percentage of {alpha}Eß7+ cells among CD4+CD25+ T cells, we found a distinct hierarchy of Nihigh > Nilow > Nivery low with regard to the frequencies of total CD4+CD25+ and CD4+CD25+{alpha}Eß7+ T cells. CD4+CD25+{alpha}Eß7+ T cells studied in vitro have been reported to have a stronger suppressive capacity than CD4+CD25+{alpha}Eß7– T cells (19, 24), and in vivo {alpha}Eß7 expression is important for CD4+CD25+ Treg cells to enter into the inflamed tissue and suppress activated T cells (25). In our experimental system, CD4+CD25+{alpha}Eß7+ T cells were more effective than CD4+CD25+{alpha}Eß7– T cells in preventing nickel hypersensitivity in Nivery low mice. Additional studies are needed to clarify whether the enhanced suppressive capacity of CD4+CD25+{alpha}Eß7+ T cells is due to a superior suppressive ability on a single-cell level, a better homing potential or a combination of the two. Consistent with published data (19, 24), those CD4+CD25+ T cells that fail to express {alpha}Eß7 probably also show some degree of suppressive capacity because we observed that removal of {alpha}Eß7+ T cells from CD4+ T cells only partially alleviated the suppression.

It is well established that oral administration of antigens favors tolerance induction, include CD4+CD25+ Treg cell differentiation (26). These CD4+CD25+ Treg cells rapidly divided preferentially in gut lymphoid tissue (27). Multiple mechanisms have been proposed to explain oral tolerance and Treg cell induction. One of them is that the intestinal mucosa has high basal levels of IL-4, IL-10 and transforming growth factor-ß (TGF-ß) (28), and short after oral antigen uptake, expression of these cytokines up-regulated (29). These cytokines, especially TGF-ß, play a central role in oral tolerance; it not only be able to convert naive peripheral CD4+CD25– T cells to CD4+CD25+ Treg cells via induction of FoxP3 (30) but also act as master regulators of most of mechanisms triggered by antigen feeding (31). Within CD4+CD25+ Treg cells, CD4+CD25+{alpha}Eß7– cells showed naive-like phenotype, CD4+CD25+{alpha}Eß7+ represents effector/memory-like phenotype and might be induced or expanded in response to antigenic stimulation (19). Interestingly, the expression of {alpha}Eß7 on CD4+CD25+ Treg cells is positively regulated by TGF-ß (25). Therefore, TGF-ß influences both the number of CD4+CD25+ Treg cells and CD4+CD25+{alpha}Eß7+ Treg cells. Although no data about TGF-ß up-regulation after oral uptake of nickel exist, it has been reported that i.p. injection of nickel enhances the TGF-ß level in rat serum (32); thus, it is conceivable that oral administration of nickel may enhance the percentages of CD4+CD25+ in CD4+ T cells and CD4+CD25+{alpha}Eß7+ T in CD4+CD25+ Treg cells.

Furthermore, nickel ions have remarkable chemical versatility, which enables them to generate a great variety of different metal–protein complexes or neoantigens, as discussed in detail elsewhere (20). Since none of the animals was primed and the only difference between the three mouse subsets was their exposure to nickel, one can indirectly conceive that an increasing nickel concentration in the body is associated with an increase in the variety of different neoantigens induced by nickel ions and, consequently, different nickel-reactive T cell clones and Treg cells. In correlation with this, the percentage of CD25+ cells among CD4+ T cells and the percentage of {alpha}Eß7+ cells among CD4+CD25+ T cells were both significantly higher in Nihigh mice when compared with Nilow mice (Fig. 8). Therefore, a higher exposure to nickel may lead to a more diverse nickel-reactive T cell repertoire and thus account for the differences in the strengths of the differentiated Treg cell populations (including the CD8+ T cells in Nihigh mice) in preventing hypersensitivity responses.

Interestingly, nickel-specific CD8+ Treg cells could be identified in Nihigh mice. A possible explanation for this could be that only in the Nihigh mice the self-proteins bound to nickel reach a high enough concentration so that sufficient numbers of nickel-induced neoantigens are presented to CD8+ T cells (33, 34). In order to be cross-presented by MHC-I molecules, extracellular antigens have to be presented at a high concentration (34–36).

By blocking CD25 on Treg cells, we could compare the suppressive power of CD4+CD25+ and CD8+ Treg cells in response to nickel. Even without CD4+CD25+ Treg cells, the remaining T cell population in Nihigh mice contained powerful suppressor T cells since they were also able to prevent sensitization induced by NiCl2/H2O2. Whether this superior suppressive capacity of CD8+ Treg cells is due to a higher frequency or a stronger suppressive ability on a single-cell level when compared with CD4+CD25+ Treg cells is unknown. Nevertheless, transfer of Nihigh CD8+ Treg cells alone cannot prevent sensitization to NiCl2/H2O2. For this, both CD4+ and CD8+ T cells have to be co-transferred. Indeed, synergism between suppressor–inducer CD4+ T cells and CD8+ suppressor–effector T cells has been observed in other models (37–42), but the mode of interaction of these cells remains poorly understood. As an example, when tolerance to trinitrophenol (TNP) was induced by i.v. injection of TNP-coupled syngeneic splenocytes, the tolerance transfer by CD8+ T suppressor–effector cells required either the co-transfer of TNP-specific CD4+ T suppressor–inducers or treatment of recipient mice with agonistic anti-CD40 mAb (43). These findings suggest that the CD8+ T suppressor–effector cells acquire some kind of help from activated CD4+ T suppressor–inducers which act through their CD40 ligand. Consistent with this, after the transfer of unsorted Nihigh T cells (containing both CD4+ and CD8+ T cells) to Nilow recipients, vigorous recipient immunization with NiCl2/H2O2 is required for the activation of CD4+ T cells and subsequent infectious spread of tolerance from donor cells to cells of the recipient (8).

CD4+ suppressor–inducer cells that cooperate with CD8+ suppressor–effector T cells in the suppression of DTH responses were characterized in detail in the model of anterior chamber-induced immune deviation (ACAID). Tolerance induction in that model (38, 41, 44) shows a remarkable number of similarities with that of Nihigh mice (16, 45). In ACAID, the CD4+ Treg suppressor–inducer cells were unequivocally identified as CD25+ T cells (41). We found that a population of Nihigh T cells, which comprised both CD8+ T cells and CD4+CD25– T cells, was able to prevent the sensitization from NiCl2/H2O2, whereas neither CD4+ T cells alone nor CD8+ T cells alone was capable of doing so. While this might suggest that Nihigh CD4+CD25– T cells acted as suppressor–inducers, we did not explicitly show that Nihigh CD4+CD25+ T cells were unable to do so. We cannot rule out that the CD4+CD25– T cells transferred along with CD8+ T cells switched their phenotype from CD25– to CD25+ in the post-transfer period, in which the recipient mice were immunized with NiCl2/H2O2 before being challenged 9 days later. Another unresolved question is why it takes the ensemble of CD8+ suppressor–effector and CD4+ suppressor–inducer T cells in order to prevent sensitization from NiCl2/H2O2, whereas CD4+CD25+ Treg cells are unable to do so but suffice to prevent the sensitization induced by NiCl2 alone.

With regard to the daily amount of nickel per kilogram body weight that is taken up from food and drinking water (12), there appears to be no fundamental difference between humans and Nilow mice, and conceivably, the same is true for the nickel-specific CD4+CD25+ Treg cells described in non-allergic humans (4) and the Nilow mice studied here. In these animals, the protection provided by CD4+CD25+ Treg cells is overcome when nickel ions are encountered in the context of danger, such as H2O2 which mimics inflammation. If we extrapolate from the Nilow mice to humans, nickel-specific CD4+CD25+ Treg cells could account for the lack of sensitization when humans encounter nickel ions on the intact skin, that is, in the absence of danger. However, when human exposure to nickel occurs in the context of danger, such as ear piercing or irritated skin, CD4+CD25+ Treg cells may be less sufficient at protecting from sensitization. Furthermore, we hypothesize that if the oral uptake of nickel in humans were as low as that of our Nivery low mice, humans might lack nickel-specific Treg cells altogether; thus, an increase in the incidence of nickel allergy would ensue.

By definition, the hypersensitivity induced in our study is a DTH rather than CHS reaction. Although CHS is one type of DTH response, it differs from other forms of DTH responses. While the effector T cells in DTH response are CD4 T cells, those in CHS response are mainly CD8 T cells, although in some instance can also be CD4 T cells. Furthermore, in DTH responses, CD4 T cells act as effector T cells whereas they exert a regulatory function in CHS responses (46, 47). In this respect, our nickel model well fits the standard of a DTH response since Nilow mice sensitized by injection of NiCl2/H2O2 harbor CD4 effector cells (15, X. Wu et al., unpublished data). By this standard, nickel allergy in humans is also not a pure CHS response since nickel-reactive T cells in human peripheral blood are mainly of the CD4+ subtype (48–50). Actually, when we further study the cause of nickel allergy in human, one can find that nickel allergy is really not pure CHS response because it can be caused by either contact nickel at irritated skin or ‘intradermal’ contact such as ear piercing, the latter is typical DTH response. Therefore, our mouse model is a useful animal model to study nickel allergy and tolerance with relevance to human nickel allergy.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
NiPERA; Deutsche Forschungsgemeinschaft (Project C8 of SFB 503).


   Acknowledgements
 
We thank our colleagues at the Institut für Umweltmedizinische Forschung for critical reading of the manuscript.


   Abbreviations
 
ACAID, anterior chamber-induced immune deviation
ANOVA, analysis of variance
APC, allophycocyanin
CHS, contact hypersensitivityDNFB2,4-dinitrofluorobenzene
DTH, delayed-type hypersensitivity
H2O2, hydrogen peroxide
i.d., intradermally
i.p., intra-peritoneally
i.v., intravenously
TGF-ß, transforming growth factor-ß
Treg, T regulatory

   Notes
 
Transmitting editor: S. Romagnani

Received 27 September 2006, accepted 17 April 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

  1. Jensen CS, Lisby S, Baadsgaard O, Byrialsen K, Menne T. Release of nickel ions from stainless steel alloys used in dental braces and their patch test reactivity in nickel-sensitive individuals. Contact Dermatitis (2003) 48:300.[CrossRef][Web of Science][Medline]
  2. Matzinger P. The danger model: a renewed sense of self. Science (2002) 296:301.[Abstract/Free Full Text]
  3. Jensen CS, Lisby S, Baadsgaard O, Volund A, Menne T. Decrease in nickel sensitization in a Danish schoolgirl population with ears pierced after implementation of a nickel-exposure regulation. Br. J. Dermatol. (2002) 146:636.[CrossRef][Web of Science][Medline]
  4. Cavani A, Nasorri F, Ottaviani C, Sebastiani S, De Pita O, Girolomoni G. Human CD25 + regulatory T cells maintain immune tolerance to nickel in healthy, nonallergic individuals. J. Immunol. (2003) 171:5760.[Abstract/Free Full Text]
  5. Smith-Sivertsen T, Tchachtchine V, Lund E. Environmental nickel pollution: does it protect against nickel allergy? J. Am. Acad. Dermatol. (2002) 46:460.[CrossRef][Web of Science][Medline]
  6. Hertel RF, Maass T, Mueller VR. Environmental health criteria 108: nickel. In: International Programme on Chemical safety s. (1991) Geneva, Switzerland: World Health Organization.
  7. Komagata Y, Weiner HL. Oral tolerance. Rev. Immunogenet. (2000) 2:61.[Medline]
  8. Artik S, Haarhuis K, Wu X, Begerow J, Gleichmann E. Tolerance to nickel: oral nickel administration induces a high frequency of anergic T cells with persistent suppressor activity. J. Immunol. (2001) 167:6794.[Abstract/Free Full Text]
  9. Ishii N, Moriguchi N, Nakajima H, Tanaka S, Amemiya F. Nickel sulfate-specific suppressor T cells induced by nickel sulfate in drinking water. J. Dermatol. Sci. (1993) 6:159.[CrossRef][Medline]
  10. van Hoogstraten IM, von Blomberg BM, Boden D, Kraal G, Scheper RJ. Effects of oral exposure to nickel or chromium on cutaneous sensitization. Curr. Probl. Dermatol. (1991) 20:237.[Medline]
  11. Mortz CG, Lauritsen JM, Bindslev-Jensen C, Andersen KE. Nickel sensitization in adolescents and association with ear piercing, use of dental braces and hand eczema. The Odense Adolescence Cohort Study on Atopic Diseases and Dermatitis (TOACS). Acta Derm. Venereol. (2002) 82:359.[CrossRef][Web of Science][Medline]
  12. Pennington JA, Jones JW. Molybdenum, nickel, cobalt, vanadium, and strontium in total diets. J. Am. Diet. Assoc. (1987) 87:1644.[Web of Science][Medline]
  13. Veien NK, Andersen MR. Nickel in Danish food. Acta Derm. Venereol. (1986) 66:502.[Web of Science][Medline]
  14. Lowenthal JW, Corthesy P, Tougne C, Lees R, MacDonald HR, Nabholz M. High and low affinity IL 2 receptors: analysis by IL 2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61. J. Immunol. (1985) 135:3988.[Abstract]
  15. Artik S, von Vultee C, Gleichmann E, Schwarz T, Griem P. Nickel allergy in mice: enhanced sensitization capacity of nickel at higher oxidation states. J. Immunol. (1999) 163:1143.[Abstract/Free Full Text]
  16. Roelofs-Haarhuis K, Wu X, Gleichmann E. Oral tolerance to nickel requires CD4(+) invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells. J. Immunol. (2004) 173:1043.[Abstract/Free Full Text]
  17. Roelofs-Haarhuis K, Wu X, Nowak M, Fang M, Artik S, Gleichmann E. Infectious nickel tolerance: a reciprocal interplay of tolerogenic APCs and T suppressor cells that is driven by immunization. J. Immunol. (2003) 171:2863.[Abstract/Free Full Text]
  18. Fehervari Z, Sakaguchi S. CD4+ Tregs and immune control. J. Clin. Invest. (2004) 114:1209.[CrossRef][Web of Science][Medline]
  19. Lehmann J, Huehn J, de la Rosa M, et al. Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25– regulatory T cells. Proc. Natl Acad. Sci. USA (2002) 99:13031.[Abstract/Free Full Text]
  20. Draeger H, Wu X, Roelofs-Haarhuis K, Gleichmann E. Nickel allergy versus nickel tolerance: can oral uptake of nickel protect from sensitization? J. Environ. Monit. (2004) 6:2.
  21. Stein-Streilein J, Streilein JW. Anterior chamber associated immune deviation (ACAID): regulation, biological relevance, and implications for therapy. Int. Rev. Immunol. (2002) 21:123.[CrossRef][Medline]
  22. Thornton AM, Shevach EM. CD4+ CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. (1998) 188:287.[Abstract/Free Full Text]
  23. Baecher-Allan C, Viglietta V, Hafler DA. Inhibition of human CD4+CD25+high regulatory T cell function. J. Immunol. (2002) 169:6210.[Abstract/Free Full Text]
  24. McHugh RS, Whitters MJ, Piccirillo CA, et al. CD4+ CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity (2002) 16:311.[CrossRef][Web of Science][Medline]
  25. Suffia I, Reckling SK, Salay G, Belkaid Y. A role for CD103 in the retention of CD4+CD25+ Treg and control of Leishmania major infection. J. Immunol. (2005) 174:5444.[Abstract/Free Full Text]
  26. Zhang X, Izikson L, Liu L, Weiner HL. Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration. J. Immunol. (2001) 167:4245.[Abstract/Free Full Text]
  27. Hauet-Broere F, Unger WW, Garssen J, Hoijer MA, Kraal G, Samsom JN. Functional CD25– and CD25+ mucosal regulatory T cells are induced in gut-draining lymphoid tissue within 48 h after oral antigen application. Eur. J. Immunol. (2003) 33:2801.[CrossRef][Web of Science][Medline]
  28. Weiner HL. Induction and mechanism of action of transforming growth factor-beta-secreting Th3 regulatory cells. Immunol. Rev. (2001) 182:207.[CrossRef][Web of Science][Medline]
  29. Gonnella PA, Chen Y, Inobe J, Komagata Y, Quartulli M, Weiner HL. In situ immune response in gut-associated lymphoid tissue (GALT) following oral antigen in TCR-transgenic mice. J. Immunol. (1998) 160:4708.[Abstract/Free Full Text]
  30. Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+CD25– naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor FoxP3. J. Exp. Med. (2003) 198:1875.[Abstract/Free Full Text]
  31. Faria AM, Weiner HL. Oral tolerance and TGF-beta-producing cells. Inflamm. Allergy Drug Targets (2006) 5:179.[Medline]
  32. Chen CY, Huang YL, Lin TH. Association between oxidative stress and cytokine production in nickel-treated rats. Arch. Biochem. Biophys. (1998) 356:127.[CrossRef][Web of Science][Medline]
  33. Kurts C, Kosaka H, Carbone FR, Miller JF, Heath WR. Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8+ T cells. J. Exp. Med. (1997) 186:239.[Abstract/Free Full Text]
  34. Heath WR, Carbone FR. Cross-presentation, dendritic cells, tolerance and immunity. Annu. Rev. Immunol. (2001) 19:47.[CrossRef][Web of Science][Medline]
  35. Kurts C, Sutherland RM, Davey G, et al. CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose. Proc. Natl Acad. Sci. USA (1999) 96:12703.[Abstract/Free Full Text]
  36. Kurts C, Miller JF, Subramaniam RM, Carbone FR, Heath WR. Major histocompatibility complex class I-restricted cross-presentation is biased towards high dose antigens and those released during cellular destruction. J. Exp. Med. (1998) 188:409.[Abstract/Free Full Text]
  37. Tan P, Anasetti C, Martin PJ, Hansen JA. Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor. J. Immunol. (1990) 145:485.[Abstract]
  38. Kosiewicz MM, Streilein JW. Intraocular injection of class II-restricted peptide induces an unexpected population of CD8 regulatory cells. J. Immunol. (1996) 157:1905.[Abstract]
  39. Li SP, Lee SI, Wang Y, Domer JE. Candida albicans mannan-specific, delayed hypersensitivity down-regulatory CD8+ cells are genetically restricted effectors and their production requires CD4 and I-A expression. Int. Arch. Allergy Immunol. (1996) 109:334.[Web of Science][Medline]
  40. Negoro T, Satoh K, Iinuma F, Tobe T, Watanabe M. Induction of CD4+ regulatory T cells by TPA in mice: contra-suppression by CD8+ T cells. Biol. Pharm. Bull. (2002) 25:172.[CrossRef][Web of Science][Medline]
  41. Skelsey ME, Mayhew E, Niederkorn JY. CD25+, interleukin-10-producing CD4+ T cells are required for suppressor cell production and immune privilege in the anterior chamber of the eye. Immunology (2003) 110:18.[CrossRef][Web of Science][Medline]
  42. Sarantopoulos S, Lu L, Cantor H. Qa-1 restriction of CD8+ suppressor T cells. J. Clin. Invest. (2004) 114:1218.[CrossRef][Web of Science][Medline]
  43. Ferguson TA, Herndon J, Elzey B, Griffith TS, Schoenberger S, Green DR. Uptake of apoptotic antigen-coupled cells by lymphoid dendritic cells and cross-priming of CD8+ T cells produce active immune unresponsiveness. J. Immunol. (2002) 168:5589.[Abstract/Free Full Text]
  44. Sonoda KH, Stein-Streilein J. Ocular immune privilege and CD1d-reactive natural killer T cells. Cornea (2002) 21:S33.[CrossRef][Medline]
  45. Nowak M, Kopp F, Roelofs-Haarhuis K, Wu X, Gleichmann E. Oral nickel tolerance: Fas ligand-expressing invariant NK T cells promote tolerance induction by eliciting apoptotic death of antigen-carrying, effete B cells. J. Immunol. (2006) 176:4581.[Abstract/Free Full Text]
  46. Grabbe S, Schwarz T. Immunoregulatory mechanisms involved in elicitation of allergic contact hypersensitivity. Immunol. Today (1998) 19:37.[CrossRef][Web of Science][Medline]
  47. Saint-Mezard P, Berard F, Dubois B, Kaiserlian D, Nicolas JF. The role of CD4+ and CD8+ T cells in contact hypersensitivity and allergic contact dermatitis. Eur. J. Dermatol. (2004) 14:131.[Web of Science][Medline]
  48. Karttunen R, Silvennoinen-Kassinen S, Juutinen K, Andersson G, Ekre HP, Karvonen J. Nickel antigen induces IL-2 secretion and IL-2 receptor expression mainly on CD4+ T cells, but no measurable gamma interferon secretion in peripheral blood mononuclear cell cultures in delayed type hypersensitivity to nickel. Clin. Exp. Immunol. (1988) 74:387.[Web of Science][Medline]
  49. Silvennoinen-Kassinen S, Ikaheimo I, Karvonen J, Kauppinen M, Kallioinen M. Mononuclear cell subsets in the nickel-allergic reaction in vitro and in vivo. J. Allergy Clin. Immunol. (1992) 89:794.[CrossRef][Web of Science][Medline]
  50. Lisby S, Hansen LH, Menn T, Baadsgaard O. Nickel-induced proliferation of both memory and naive T cells in patch test-negative individuals. Clin. Exp. Immunol. (1999) 117:217.[CrossRef][Web of Science][Medline]

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