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The Journal of Immunology, 2004, 173: 1043-1050.
Copyright © 2004 by The American Association of Immunologists

Oral Tolerance to Nickel Requires CD4+ Invariant NKT Cells for the Infectious Spread of Tolerance and the Induction of Specific Regulatory T Cells1

Karin Roelofs-Haarhuis, Xianzhu Wu and Ernst Gleichmann2

Institut für Umweltmedizinische Forschung (IUF), Heinrich Heine University, Düsseldorf, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously, oral administration of nickel to C57BL/6 wild-type (WT) mice was shown to render both their splenic T cells and APCs (i.e., T cell-depleted spleen cells) capable of transferring nickel tolerance to naive syngeneic recipients. Moreover, sequential adoptive transfer experiments revealed that on transfer of tolerogenic APCs and immunization, the naive T cells of the recipients differentiated into regulatory T (Treg) cells. Here, we demonstrate that after oral nickel treatment J{alpha}18–/– mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those J{alpha}18–/– mice did tolerize WT recipients. Hence, during oral nickel administration, tolerogenic APCs are generated that require iNKT cell help for the induction of Treg cells. To obtain this help, the tolerogenic APCs must address the iNKT cells in a CD1-restricted manner. When J{alpha}18–/– mice were used as recipients of cells from orally tolerized WT donors, the WT Treg cells transferred the tolerance, whereas WT APCs failed to do so, although they proved tolerogenic on transfer to WT recipients. However, J{alpha}18–/– recipients did become susceptible to the tolerogenicity of transferred WT APCs when they were reconstituted with IL-4- and IL-10-producing CD4+ iNKT cells. We conclude that CD4+ iNKT cells are required for the induction of oral nickel tolerance and, in particular, for the infectious spread of tolerance from APCs to T cells. Once induced, these Treg cells, however, can act independently of iNKT cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The NKT cells have been identified as a heterogeneous lymphoid lineage that is distinct from conventional T cells and NK cells because they express both a TCR and the NK cell marker NK1.1 (1). The majority of NKT cells express an invariant TCR which in mice is encoded by the V{alpha}14 and J{alpha}18 (formerly J{alpha}281) gene segment paired with a limited TCR {beta}-chain repertoire (2). These invariant NKT (iNKT)3 cells recognize glycolipids in a CD1d-restricted manner (3, 4). CD1 molecules are nonpolymorphic, MHC class I-like surface proteins that are constitutively expressed on many lymphohemopoietic cell types, including dendritic cells (DCs) and B cells from the splenic marginal zone (1, 5). To date only a few iNKT cell-activating glycolipids have been identified (6). Examples of these include the well studied {alpha}-galactosylceramide, a component of a marine sponge (7), and the disialoganglioside GD3, a cell membrane component in humans and mice (4).

The importance of CD1-restricted iNKT cells in promoting peripheral T cell tolerance was suggested after observing the reduced frequency and function of these cells in patients with autoimmune diseases (8, 9). The pathogenic significance of this association was then experimentally proved by demonstrating that the spontaneous development of autoimmune diseases could be prevented by either overexpression (10) or in vivo activation of iNKT cells (11), or their adoptive transfer to iNKT cell-deficient mice (12). Furthermore, CD1-reactive mouse iNKT cells are required for survival of rat islet xenografts in mice (13), and for the induction of anterior chamber-associated immune deviation (ACAID) (14). Moreover, CD1-reactive NKT cells were also required for the commencement of specific suppression of delayed-type hypersensitivity (DTH) induced by Ag administration via the oral route or the portal vein (15). Although the oral administration of Ag is an established protocol leading to peripheral T cell tolerance, its underlying mechanisms remain elusive (16). A consequence of orally induced T cell tolerance, and thus a possible mechanism, is the reported development of regulatory T (Treg) cells after high doses of tolerogen (17, 18). Upon TCR engagement, these Treg cells were shown to secrete large amounts of IL-4, IL-10, and TGF-{beta}1, cytokines renowned for their ability to down-regulate Th1 responses (17). For mechanisms in the immediate stages of tolerance induction, possible candidates include the rapid cytokine release from activated iNKT cells. These cytokines are now considered to modulate immune responses by shifting the balance between Th1 and Th2 (19). This iNKT cell-mediated modulation of the Th1/Th2 balance could, in turn, depend on the mode or duration of iNKT cell activation. For example, whereas a single injection of {alpha}-galactosylceramide was found to induce a Th1 response (20), multiple injections were observed to promote a Th2 response (11). Alternatively, other studies suggest that it is the maturation state of the APCs during their interaction with iNKT cells that is the critical aspect for the iNKT cell-mediated regulation of the Th1-Th2 balance (21, 22, 23). Further, it has been shown that in contrast to CD40-activated DCs, which polarized iNKT cells to secrete Th1-type cytokines, blockade of CD86 costimulation resulted in a shift toward Th2-type cytokines (22, 23).

Our previous work has shown that oral administration of Ni2+ ions, as present in NiCl2 and NiSO4, resulted in a long-lasting immunological tolerance toward the neo-Ags generated by the contact allergen nickel (24). Adoptive transfer of 102 bulk T cells or 102 splenic APCs (T cell-depleted spleen cells) from orally tolerized C57BL/6 wild-type (WT) donors into naive syngeneic recipients protected the latter from sensitization to nickel; sensitization in this model is induced by the coadministration of NiCl2 and H2O2 as an adjuvant (24, 25, 26). The fact that such a small number of T cells or APCs, which contained mainly B cells, from the orally tolerized donors succeeded to tolerize naive recipient mice was further demonstrated to occur via infectious tolerance. In short, after adoptive cell transfer, we observed that tolerance did indeed infectiously spread from the donor T cells and the donor APCs to the APCs and T cells of naive recipients, respectively (25). Thus, although infectious tolerance represents an impressive amplification loop in the development of peripheral T cell tolerance, the cellular and molecular mechanisms involved are far from resolved (27).

In the present study, we now observe that iNKT cells are required not only for the oral induction of nickel tolerance but also for its infectious spread from tolerogenic donor APCs to the naive T cells of the recipients. However, once the oral nickel administration to WT donor mice induced Treg cells specific for nickel, the suppressive abilities of these cells was largely independent from the presence of iNKT cells in the recipients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free female C57BL/6J WT mice, which express Ly5.2 (CD45.2), were obtained from Janvier (Le Genest St. Isle, France). Congenic Ly5.1+ (CD45.1+) C57BL/6J (B6.SJL-PtPrca Pep3b/BoyJ), IL4–/– (C57BL/6-IL-4tm1Nnt), IL-10–/– (C57BL/6-IL-10tm1Cgn), and IFN-{gamma}–/– (C57BL/6-Infgtm1Ts) females were purchased from The Jackson Laboratory (Bar Harbor, ME). J{alpha}18–/– mice were created at Chiba University (Chuoku Chiba, Japan) and backcrossed nine times with C57BL/6 mice (28); they were a gift from Dr. Balk (Beth Israel Deaconess Medical Center, Harvard University, Boston, MA). CD1–/– mice were bred at Beth Israel Deaconess Medical Center and backcrossed six times with C57BL/6 mice (29); they were a gift from Dr. S. Kaufmann (Max-Planck-Institute of Infection Biology, Berlin, Germany). Mice received food and water ad libitum, as described (24, 26), and were 6–14 wk old at the onset of experiments.

Reagents

NiCl2·6H2O (hereafter referred to as NiCl2) was purchased from Sigma-Aldrich Chemie (Steinheim, Germany), and H2O2 was obtained from E. Merck (Darmstadt, Germany).

Antibodies

The following anti-mouse mAb were purchased from BD PharMingen (Heidelberg, Germany): APC-labeled anti-CD3{epsilon} (clone 145-2C11); PE- or PerCP-labeled anti-CD4 (clone RM4-5); PE-labeled anti-CD8{beta}.2 (clone 53-5.8), APC-labeled anti-CD11c (clone HL3); FITC-labeled anti-CD19 (clone 1D3); APC-labeled anti-B220 (clone 30-F11); PE-labeled anti-CD45.1 (clone A20); FITC-labeled anti-CD45.2 (clone 104); FITC-labeled anti-I-Ab (clone AF6-120.1); PE- or PerCP-labeled anti-NK1.1 (clone PK136); and FITC-labeled anti-TCR{beta}-chain (clone H57-597). Magnetically labeled anti-CD4, anti-CD11c, anti-CD19, anti-CD90, anti-B220, anti-MHC-II, anti-FITC, and anti-PE mAb were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Oral tolerance induction

Mice were treated with 10 mM NiCl2 in the drinking water (24) for at least 4 up to a maximum of 8 wk. They were continuously treated until sacrificed for removal of their spleen. Age- and sex-matched control mice received tap water without additional NiCl2.

Immunization of mice

Mice were immunized as described previously (24, 26). In the case of Ni, mice were primed by intradermal (i.d.) injection into both flanks (50 µl each) with either 10 mM NiCl2 in sterile, pyrogen-free saline or 10 mM NiCl2 in saline containing 1% H2O2 (NiCl2/H2O2). In the case of DNFB, mice were primed by painting 0.5% (w/v) DNFB on the shaved flanks (25 µl each); DNFB was resolved in acetone-olive oil (4:1, v/v).

Challenge for recall and ear swelling test

Ten days after immunization, mice were rechallenged by injecting 50 µl of 10 mM NiCl2 in sterile, pyrogen-free saline into the pinna of each ear. Forty-eight hours after rechallenge with NiCl2, DTH reactions were determined by measuring the increment in ear thickness in comparison with the prechallenge values. To determine the prechallenge values, mice were anesthetized with diethyl ether, whereas for the measurement after challenge, mice were killed by asphyxiation with CO2. Measurements were performed in a blind manner (24, 26), using a micrometer gauge (Oditest D 1000 gauge; Dyer, Lancaster, PA). The results are shown as the mean ear swelling response from groups of 5–6 mice and are expressed in units of millimeters x 10–2 + SE.

Sorting of T cells and APCs for adoptive transfer studies

For the transfer of T cells, single-cell suspensions of erythrocyte-free spleen cells, which contained 30–35% T cells, 60–65% B cells, and 1–2% DC, were passed through nylon wool columns, and then depleted of CD11c+, CD19+, and MHC-II+ cells using a magnetic cell sorter (autoMACS; Miltenyi Biotec). To transfer APCs, single-cell suspensions of erythrocyte-free spleen cells were depleted of CD4+, CD8+, and CD90+ T cells using the autoMACS. The remaining cells, containing 90–95% CD19+MHC-II+ B cells and 1–3% CD11c+MHC-IIhigh DC, will be referred to as APCs. The purity of the resulting T cell and APC fractions was determined by FACS and were found to be contaminated with <0.5% CD19+MHC-II+ and CD11c+MHC-IIhigh APCs and <0.5% CD4+CD3+ and CD8+CD3+ T cells, respectively. In some experiments, purified T cells or total spleen cells were depleted of NK1.1+ cells or CD4+ cells by using PE-conjugated anti-NK1.1 mAb and PE-conjugated anti-CD4 mAb, respectively, and then counterstained with anti-PE MicroBeads. After the stained cells were applied to the MACS, the negative fractions contained <0.1% TCR{beta}+NK1.1+ cells and CD3+CD4+ T cells, respectively.

For sequential adoptive transfer experiments on day 0, APCs of primary, orally tolerized donors were transferred to a first set of recipients differing in Ly5. On day 1, these recipients were immunized with NiCl2/H2O2 and were used 10 days later as donors of T cells to be transferred to a second set of recipients. The T cell fraction obtained from the first set of recipients (or the secondary donors) went through an additional depletion step which was based on the difference in the markers Ly5.1 and Ly5.2 between the primary donors and the first set of recipients (25). This depletion step excluded the possibility that the tolerance induction in the second set of recipients was simply due to contamination by tolerogenic APCs that were carried over from the primary donors. After this depletion, <0.1% of the T cells transferred originated from the primary donors.

Adoptive cell transfers

Cell suspensions, containing the type and the number of cells indicated, were diluted in sterile, pyrogen-free PBS and injected i.v. into recipient mice (150 µl). One day later, mice were immunized with NiCl2/H2O2 or the control compounds indicated. Ten days thereafter, the recipients were rechallenged at the ears; 48 h later, their ear swelling response was measured. An exception to this protocol was the first set of recipients in the experiments on infectious tolerance, because these animals were not rechallenged after the immunization. Unless otherwise mentioned, each group of mice evaluated in the ear swelling test consisted of five animals.

Statistical analysis

Statistical significances were determined by ANOVA followed by the Newman-Keuls test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
iNKT cells are required for the induction of oral tolerance toward nickel, but not for nickel sensitization

As previously reported (26), i.d. injection of NiCl2 and H2O2 sensitizes C57BL/6 WT mice to nickel, so that after rechallenge with NiCl2 in the ears they exhibit an increased ear swelling response (Fig. 1, bar 1). J{alpha}18–/– mice, which lack iNKT cells (30), also developed an increased ear swelling after immunization with NiCl2/H2O2 and rechallenge with NiCl2, the magnitude of this response being comparable with that of equally treated WT mice (Fig. 1, bars 3 and 1, respectively). Hence, iNKT cells are not required for either de novo sensitization or specific recall of nickel hypersensitivity in vivo. As also shown previously (24), the ear swelling in WT mice immunized with NiCl2/H2O2 and challenged with NiCl2 that had received 10 mM NiCl2 in the drinking water for 4 wk before sensitization was as low as the background ear swelling previously observed in mice that were not immunized, but only challenged with NiCl2 (26). The background response in the WT mice indicates the development of tolerance (Fig. 1, bar 2). In contrast, orally treated J{alpha}18–/– mice showed a high ear swelling after immunization with NiCl2/H2O2 and rechallenge with NiCl2 (Fig. 1, bar 2 vs bar 4), indicating that their lack of iNKT cells rendered them insensitive to the induction of oral tolerance toward nickel. Orally treated J{alpha}18–/– mice that were immunized with NiCl2 in the absence of H2O2 only showed a background response (Fig. 1, bar 5). The tolerance induced in J{alpha}18–/– mice by oral administration of NiCl2 was specific for nickel ions, because these mice showed a normal immune response to DNFB (Fig. 1, bar 6).



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  		FIGURE 1. In contrast to WT mice, J{alpha}18–/– mice fail to develop nickel tolerance after a 4-wk course of 10 mM NiCl2 in the drinking water. WT and J{alpha}18–/– mice received 10 mM NiCl2 in the drinking water for a period of 4 wk or remained untreated. After immunization with NiCl2/H2O2, and challenge for recall with NiCl2 at the ears, their ear swelling response was determined. As negative control, J{alpha}18–/– mice, which were orally treated with NiCl2, received i.d. injections of NiCl2, instead of NiCl2/H2O2 at the flanks (bar 5). To control the specificity, J{alpha}18–/– mice, which were orally either treated with NiCl2 or not, were immunized and rechallenged with DNFB (bars 6 and 7); untreated J{alpha}18–/– mice which were only challenged with DNFB served as background control (bar 8). In this and the subsequent figures, the asterisks indicate a significant difference (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001) between the groups compared by brackets. Results represent one of two experiments that yielded comparable results. In this and the following figures, white bars indicate the critical experimental groups. Bar numbers mentioned in the text refer to the bars read from top to bottom.

 
iNKT cell-deficient mice as donors: iNKT cells are required for the induction of nickel-specific Treg cells, but not for the generation of tolerogenic APCs

In view of the resistance of J{alpha}18–/– mice to tolerization, we investigated whether both their T cells and APCs failed to be affected in a tolerogenic way or whether only one of the two cell populations failed to respond to the oral tolerance treatment. To address this question, adoptive transfer experiments were performed in which each cell population was tested separately (24, 25). Either 104 splenic T cells or APCs (i.e., T cell-depleted spleen cells) from J{alpha}18–/– donors, which had been orally treated with 10 mM NiCl2 for 4 wk, were transferred to naive WT recipients; after cell transfer, the latter were immunized with NiCl2/H2O2 at the flanks and rechallenged with NiCl2 at the ears (Fig. 2). In contrast to the T cells from orally treated WT donors, those from orally treated J{alpha}18–/– donors failed to tolerize naive WT recipients as the latter showed an increased ear swelling response (Fig. 2, bars 2 and 4). Interestingly however, the APCs from orally treated J{alpha}18–/– donors did induce tolerance on transfer to naive WT recipients (Fig. 2, bar 6). Hence, although iNKT cell-deficient mice failed to be tolerized (Fig. 1) and were unable to generate the nickel-specific Treg cells, they did possess tolerogenic APCs after this treatment. Thus, iNKT cells are required for the induction of nickel-specific Treg cells, but not for that of tolerogenic APCs.



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  		FIGURE 2. J{alpha}18–/– mice possess tolerogenic APCs after the oral administration of nickel, but lack Treg cells. Prospective WT and J{alpha}18–/– donor mice were orally treated with NiCl2 or remained untreated. Splenic T cells or APCs, i.e., T cell-depleted spleen cells, from these donors were injected i.v. into naive syngeneic WT recipients (104 cells/recipient). The recipients were immunized, and their ear swelling responses were determined, as described in the legend to Fig. 1. Results represent one of three experiments that yielded comparable results.

 
iNKT cell-deficient mice as recipients: iNKT cells are nonessential for the suppressor-effector function of nickel-specific Treg cells

To test whether iNKT cells have an essential role in the effector phase of T cell-mediated suppression, 104 T cells from orally tolerized WT donors were transferred into naive J{alpha}18–/– recipients, followed by the usual experimental protocol of immunization with NiCl2/H2O2, rechallenge with NiCl2 at the ears, and measurement of the ear swelling response (Fig. 3). The successful transfer of tolerance by unseparated spleen cells from orally treated, but not from untreated WT donors, indicates that the lack of iNKT cells in the recipients did not prevent the induction of tolerance in the latter (Fig. 3, bars 1 and 2). We then separated WT spleens into T cells, which contain NKT cells, and APCs, which lack NKT cells, and used the separated cell populations as donor cells. Interestingly, 104 splenic T cells from orally tolerized WT donors were capable of transferring the tolerance into J{alpha}18–/– recipients, whereas 104 APCs from the same donors proved unable to do so (Fig. 3, bars 4 and 5).



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  		FIGURE 3. J{alpha}18–/– mice can be tolerized by the transfer of Treg cells, but not of tolerogenic APCs, obtained from orally tolerized WT donors. Prospective WT donor mice were orally tolerized or remained untreated. Spleen cells, splenic T cells, or APCs from these tolerant or naive WT donors were injected i.v. into naive J{alpha}18–/– recipients (104 cells/recipient). Twenty-four hours after the transfer, all recipients were immunized, and their ear swelling responses were determined, as described in the legend to Fig. 1. Results represent one of three experiments that yielded comparable results.

 
However, in contrast to the previously reported tolerance transfer by as few as 102 T cells from orally tolerized WT donors to WT recipients (31), tolerance could be transferred to J{alpha}18–/– mice only with 104 but not with 102 or 103 T cells from orally tolerized WT donors (Fig. 4, bars 1, 2 and 3). A possible explanation for the higher number of Treg cells needed to transfer tolerance to J{alpha}18–/– recipients could be the need for the presence of a small number of iNKT cells. Conceivably, these were supplied as a small fraction of iNKT cells present among the WT donor Treg cells that were used to transfer nickel tolerance to the J{alpha}18–/– recipients. If this were correct, then the transfer of WT Treg cells depleted of contaminating iNKT cells should no longer be able to tolerize the J{alpha}18–/– recipients. However, 104 WT donor Treg cells depleted of NK1.1+ cells were still able to transfer the tolerance to J{alpha}18–/– recipients (Fig. 4, bar 5), and, in fact, the dose-response curves obtained with undepleted T cells and those depleted of NK1.1+ T cells were quite similar (Fig. 4, bars 1–4 and 5–8). These results indicate that iNKT cells are not required for the suppressor-effector functions of Treg cells that arise in WT mice orally tolerized to nickel. Apparently, once the Treg cells have been generated (in an iNKT cell-dependent fashion; Fig. 2), they are able to perform their actions in the absence of iNKT cells. However, why 103 and 102 WT donor Treg cells, depleted of NK1.1+ cells or not, did not suffice to transfer the tolerance to the J{alpha}18–/– recipients (Fig. 4, bars 2, 3, 6, and 7), remains unresolved.



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  		FIGURE 4. iNKT cells are not essential for the functional activity of Treg cells that were generated in mice orally tolerized to nickel. Prospective WT donor mice were orally tolerized or remained untreated. Indicated numbers of splenic T cells, or splenic T cells depleted of NK1.1+ cells from tolerant or naive WT donors were injected i.v. into naive J{alpha}18–/– recipients. Twenty-four hours after the transfer, the recipients were immunized, and their ear swelling responses were determined, as described in the legend to Fig. 1. Results represent one of three experiments that yielded comparable results.

 
Sequential adoptive cell transfers: iNKT cells enable the tolerogenic donor APCs to tolerize naive T cells and induce specific Treg cells

Whereas APCs can acquire tolerogenicity in the absence of iNKT cells (Fig. 2), the transfer of 104 tolerogenic APCs from WT donors failed to induce tolerance in iNKT cell-deficient recipients (Fig. 3). Conceivably, the tolerogenic donor APCs need iNKT cells as an additional cell type to allow them to convey the tolerogenic signals to the T cells of the recipient and induce new Treg cells. To test this hypothesis, infectious tolerance experiments involving two consecutive cell transfers were performed (Fig. 5). In previous experiments with WT mice (25), APCs from donors orally tolerized toward nickel were adoptively transferred into a first set of recipients. One day later, the latter were immunized with NiCl2/H2O2, and 10 days thereafter their T cells, depleted of cells from the primary donors, were transferred to a second set of recipients; this was followed by the usual experimental protocol of recipient immunization, rechallenge, and measurement of the ear swelling response. In this way, we have previously established that APCs (from orally tolerized Ly5.1+ congenic donors) can infectiously spread the tolerance to naive T cells (of WT recipients possessing Ly5.2) and thus enable them to prevent sensitization of the second set of Ly5.2+ recipients (25). Here, we aimed to elucidate why transfer of APCs from J{alpha}18–/– (Ly5.2+) donors, which were orally treated with nickel, not only induced systemic tolerance in WT recipients (Fig. 2) but also are able to render their T cells suppressive. Therefore, we adapted the experimental design so that the cells of the primary J{alpha}18–/– donors were eliminated from those of the first set of recipients before the second transfer. Because the J{alpha}18–/– mice used as the first donors are Ly5.2+, we used congenic Ly5.1+ C57BL/6 mice, instead of the usual Ly5.2+ C57BL/6 WT mice, as the first set of recipients; WT mice served as the second set of recipients (Fig. 5A). The obtained results showed that T cells from those first recipients, which possessed iNKT cells, acquired suppressive properties from the APCs of the orally treated primary donors, irrespective of whether the donor mice were iNKT cell-deficient or not (Fig. 5A, bars 2 and 4). Hence, in the presence of iNKT cells in the first recipients, not only the APCs from orally tolerized WT donors, which were iNKT cell-sufficient, but also those from orally treated J{alpha}18–/– donors could infectiously spread the tolerance to T cells of naive recipient mice. Thus, the APCs of orally treated J{alpha}18–/– mice were capable of inducing Treg cells, notwithstanding the fact that the orally treated J{alpha}18–/– mice themselves failed to develop systemic tolerance toward nickel (Fig. 1) and their T cells failed to be suppressive (Fig. 2).



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  		FIGURE 5. Tolerogenic donor APCs require iNKT cells to spread the tolerance to naive T cells and render them suppressive. A, Prospective Ly5.2+ WT (bars 1 and 2) and Ly5.2+ J{alpha}18–/– (bars 3 and 4) donor mice were orally treated with nickel or remained untreated. Thereafter, APCs from the different Ly5.2+ primary donors (104 cells/recipient) were transferred to a first set of congenic Ly5.1+ recipients. B, Prospective Ly5.1+ congenic donor mice were orally treated with nickel or remained untreated. Thereafter, APCs from the Ly5.1+ primary donors (104 cells/recipient) were transferred into a first set of Ly5.2+ WT (bars 1 and 2) or J{alpha}18–/– (bars 3 and 4) recipients. In both A and B, all recipients were immunized with NiCl2/H2O2 within 24 h after transfer. On day 11, T cells from the first set of recipients were isolated, depleted of primary donor cells, and subsequently transferred to a second set of Ly5.2+ WT recipients. The latter were immunized and rechallenged, and their ear swelling responses were determined in accordance with the standard protocol. The displayed data represent the mean ear swelling responses + SE from groups of 10 mice each. Results represent one of three experiments, which yielded comparable results.

 
Because APCs from orally tolerized WT donors failed to spread the tolerance to naive T cells in J{alpha}18–/– recipients (Fig. 3), we further investigated whether this inability to spread the tolerance was due to the iNKT cell-deficient environment. For these experiments, we reverted back to congenic Ly5.1+ C57BL/6 mice as donors and used both Ly5.2+ WT mice (Fig. 5B, bars 1 and 2), and Ly5.2+ J{alpha}18–/– mice as the first recipients (Fig. 5B, bars 3 and 4). As can be seen, if the first recipients were devoid of iNKT cells, transfer of the tolerogenic APCs failed to render their T cells suppressive (Fig. 5B, bar 4). This is evident from the fact that adoptive transfer of T cells from J{alpha}18–/– first recipients, which had received APCs from orally tolerized Ly5.1+ donors, to the second set of recipients failed to prevent sensitization of the latter. We conclude that iNKT cells are indeed required as intermediates for the infectious spread of tolerance from tolerogenic APCs to naive T cells in that they promote differentiation of naive T cells into Treg cells.

CD1-deficient mice as donors: tolerogenic APCs need to address iNKT cells in a CD1-restricted manner to induce tolerance in WT recipients

With the now known relevance of CD1-restricted iNKT cells in tolerance, we investigated whether tolerogenic donor APCs must cooperate with the iNKT cells of the recipients in a CD1-restricted manner to induce nickel tolerance. Therefore, APCs from orally treated CD1–/– mice were adoptively transferred to WT recipients. Because CD1–/– mice in addition to CD1 also lack iNKT cells, they failed, as expected, to be tolerized by oral treatment with 10 mM NiCl2 for 4 wk (data not shown). Hence, in this respect, they were comparable with orally treated J{alpha}18–/– mice (Fig. 1). However, differences did emerge when APCs from these orally treated J{alpha}18–/– and CD1–/– donors were adoptively transferred into WT recipients (Fig. 6). Whereas the APCs of orally treated J{alpha}18–/– donors were able to induce tolerance (Fig. 6, bar 4), the APCs of orally treated CD1–/– donors proved unable to do so (Fig. 6, bar 6). Hence, it appears that transferred tolerogenic APCs must activate the iNKT cells of the recipients in a CD1-restricted fashion to induce tolerance in WT recipients.



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  		FIGURE 6. Tolerogenic APCs need to address iNKT cells in a CD1-restricted manner to induce tolerance in WT recipients. Prospective WT, J{alpha}18–/–, and CD1–/– donor mice were orally treated with NiCl2 or were left untreated. Splenic APCs, i.e., T cell-depleted spleen cells, from these donors were injected i.v. into naive WT recipients (105 cells/recipient). Twenty-four hours after the transfer, the recipients were immunized, and their ear swelling response was determined as described in the legend to Fig. 1. Results represent one of two experiments that yielded comparable results.

 
NKT cell-deficient mice as recipients: reconstitution with CD4+ iNKT cells renders J{alpha}18–/– recipients susceptible to tolerance induction by transfer of APCs from orally tolerized WT donors

Because tolerogenic APCs appear to require CD1-restricted iNKT cells to infectiously spread the tolerance to T cells, we asked whether tolerogenic APCs would be able to tolerize J{alpha}18–/– recipients when the latter were reconstituted with iNKT cells. As a source of iNKT cells, splenocytes (107) from naive WT donors were used (Fig. 7A). As expected, the transfer of naive spleen cells alone failed to tolerize the J{alpha}18–/– recipients, as did the transfer of 105 tolerogenic APCs from orally tolerized WT donors (Fig. 7A, bars 1 and 3). However, cotransfer of tolerogenic APCs and iNKT cell-containing spleen cells from naive WT donors enabled the tolerogenic APCs to exert their tolerogenic function in the J{alpha}18–/– recipients (Fig. 7A, bar 4). The results shown in Fig. 7B demonstrate that the iNKT cells within the naive WT spleens used to reconstitute the J{alpha}18–/– recipients were, indeed, the missing cell population. To be more exact, tolerogenic WT APCs failed to induce tolerance in J{alpha}18–/– recipients when they were cotransferred with naive spleen cells from J{alpha}18–/– donors (Fig. 7B, bar 3) or with either spleen cells from naive WT donors after the depletion of NK1.1+ cells or CD4+ cells (Fig. 7B, bars 4 and 5). Therefore, the data suggest that reconstitution with CD4+ iNKT cells enables J{alpha}18–/– recipients to become tolerized after transfer of tolerogenic APCs from WT donors.



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  		FIGURE 7. Cotransfer of tolerogenic WT APCs together with naive WT spleen cells, which contain iNKT cells, induces tolerance in J{alpha}18–/– recipients. Prospective WT donor mice were orally tolerized or were left untreated, and naive J{alpha}18–/– mice served as recipients. A, J{alpha}18–/– mice were injected i.v. with total spleen cells obtained from tolerized or naive WT donors (bars 1, 2 and 4) and/or splenic APCs from tolerized or naive WT donors (bars 3–5). B, All J{alpha}18–/– mice were injected i.v. with splenic APCs from tolerized WT donors. In addition, they received either total spleen cells obtained from naive WT donors (bar 2), total spleen cells obtained from naive J{alpha}18–/– donors (bar 3), or naive WT spleen cells depleted of NK1.1+ or CD4+ cells (bars 4 and 5). In both A and B, both cell fractions were mixed before the cotransfer. Twenty-four hours after the transfer, the recipients were immunized, and their ear swelling response was determined, as described in the legend to Fig. 1. Results represent one of three experiments that yielded comparable results.

 
iNKT cell-deficient mice as recipients: IL-4- and IL-10-producing iNKT cells aid tolerogenic APCs to spread tolerance

J{alpha}18–/– recipients can be tolerized by the transfer of tolerogenic WT APCs and simultaneous reconstitution with iNKT cells (Fig. 7). Therefore, the significance of cytokines secreted by those iNKT cells was analyzed. For this purpose, experiments were conducted in which tolerogenic WT APCs were transferred with naive iNKT cell-containing spleen cells from IL-4–/–, IL-10–/–, or IFN-{gamma}–/– donors into J{alpha}18–/– recipients (Fig. 8). The percentage of T cells (both CD3+CD4+ and CD3+CD8+), NKT cells (NK1.1+TCR{beta}+), B cells (B220+MHCII+), and DCs (CD11c+MHCIIhigh) among the splenocytes of the three gene-defective strains were virtually identical with those detected in the splenocytes of WT mice, as determined by flow cytometry (data not shown). In contrast to the cotransfer of tolerogenic APCs and spleen cells from IFN-{gamma}–/– donors, which did transfer tolerance (Fig. 8, bar 5), the cotransfer of tolerogenic APCs with spleen cells from either IL-4–/– or IL-10–/– donors failed to do so (Fig. 8, bars 3 and 4). Thus, tolerogenic APCs appear to depend on IL-4- and IL-10-producing, but not IFN-{gamma}-producing iNKT cells to spread the nickel tolerance to naive T cells.



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  		FIGURE 8. Tolerogenic APCs require IL-4- and IL-10-producing iNKT cells to induce tolerance in Ja18–/– recipients. Prospective WT donor mice were orally tolerized or were left untreated. Splenic APCs (105 cells/recipient) from tolerant WT donors and spleen cells (107 cells/recipient) from naive WT, IL-4–/–, IL-10–/–, or IFN-{gamma}–/– donors were isolated, mixed, and injected i.v. into naive J{alpha}18–/– recipients. Twenty-four hours after the transfer, the recipients were immunized, and their ear swelling response was determined as described in the legend to Fig. 1. Results represent one of three experiments that yielded comparable results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study demonstrate that iNKT cells are required for the induction of specific Treg cells in both experimental systems studied here, namely oral administration of NiCl2 (no adoptive transfer involved) and the infectious tolerance spread on adoptive transfer in which tolerogenic donor APCs induce specific Treg cells in the recipients. The iNKT cells promoting the infectious tolerance were CD4+- and CD1d-restricted and had to be capable of producing IL-4 and IL-10, but not IFN-{gamma}. However, iNKT cells were not necessary for either the induction of DTH to nickel, the generation of tolerogenic APCs, or the tolerance transfer mediated by the fully functional Treg cells generated during the oral tolerization of WT donors.

The actual where, when, and how iNKT cells interact with other immune cells in the induction of oral tolerance and Treg cells is still unknown. One line of thought proposes that the liver could function as a second security line after the intestine, to ensure tolerance toward Ags that are orally ingested (32), and indeed, hepatic NKT cells were shown to be required for the induction of oral tolerance in TNBS-induced colitis, a Th1-mediated disease (33, 34). Furthermore, oral administration of colitis-extracted proteins, without an exogenous adjuvant, was shown not only to increase the number and cytolytic function of these NKT cells but also to shift their Th1-cytokine response to a Th2-type response (35, 36). In vivo, the depletion of NK1.1+ cells can prevent this shift and subsequently prevent the ability to induce tolerance (37). Thus, hepatic NKT cells can polarize an orally induced immune response to Ags, which lack costimulatory capacity, toward a Th2 response. However, other authors (32) who induced oral tolerance by a single, high dose of OVA reported that iNKT cells were dispensable for the induction of oral tolerance.

Even though J{alpha}18–/– mice failed to develop tolerance and generate specific Treg cells on oral nickel administration, the generation of tolerogenic APCs was not impaired in these iNKT cell-deficient animals. However, these APCs required signals from iNKT cells to generate Treg cells, and these findings are comparable with those observed in the ACAID model. In ACAID, intraocular CD1+F4/80+ APCs were found to acquire their tolerogenic capacity through contact with immunosuppressive factors, such as TGF-{beta}, that are present in the intraocular fluid (38). Special features of those tolerogenic APCs are a reduced expression of CD40, up-regulation of CD1d, a reduced capacity to produce the Th1-inducing cytokine IL-12, and an autocrine production of TGF-{beta}. After capturing Ag, the tolerogenic, Ag-transporting APCs were shown to migrate from the anterior chamber to the splenic marginal zone. There, they produced a variety of chemokines and formed clusters with CD1+ B cells, CD1d-reactive CD4+ iNKT cells, and CD8+ T cells (39, 40). From these results a hypothesis emerged that in these cell clusters, IL-10-producing CD4+ iNKT cells together with the tolerogenic CD1+ APCs, induced the Ag-specific CD8+ Treg cells which were shown to mediate the suppression of DTH responses in ACAID (41).

The number of common features in the ACAID and our model of oral nickel tolerance can be extended even further. In both models, the tolerogenic APCs were found to have reduced expression of CD40 (25, 42). iNKT cell production of IFN-{gamma}, which polarizes the Th1-Th2 balance toward Th1, requires APCs to produce IL-12 in a CD40-dependent manner (23). Therefore, in our experiments, it is conceivable to assume that the reduced CD40 expression by the transferred tolerogenic donor APCs shifted the cytokine production by iNKT cells toward Th2 and thus prevented the induction of Th1-mediated nickel hypersensitivity in the recipients.

In the ACAID model, transferred tolerogenic splenic B cells indirectly suppressed the recipients’ DTH reaction via the induction of specific Treg cells (43). Similarly, in orally induced nickel tolerance, splenic APCs and even the B cells alone isolated from those APCs were able to transfer the tolerance (23). We now show that APCs from orally treated donors must cooperate with iNKT cells to induce Treg cells (Fig. 5). Therefore, iNKT cells are required for mediating the infectious spread of tolerance from tolerogenic APCs to naive T cells. This conclusion is further corroborated by the results of the reverse experimental approach, showing that the APCs from orally treated J{alpha}18–/– donors succeeded in tolerizing the iNKT cell-containing WT recipients (Fig. 2); there, in the presence of iNKT cells, they were able to induce a new generation of Treg cells (Fig. 5).

There are two distinguishable subpopulations of iNKT cells, CD4+ and CD4–CD8– (double-negative) iNKT cells. In humans, the double-negative iNKT cells selectively produce Th1-type cytokines, whereas the CD4+ iNKT cells can produce both Th1- and Th2-type cytokines including IL-10 (44, 45). Whereas most studies report a regulatory role of NKT cells in human autoimmune diseases (46), Lee et al. (47) failed to find differences in CD4+ iNKT cell frequency and IL-4 production between patients suffering from insulin-dependent diabetes and healthy controls. It should be noted, however, that in the human studies only surface markers of and cytokine production by NKT cells were investigated. In contrast, the functional role of NKT cells in T cell tolerance has been well established in different animal models (10, 11, 12, 13, 14, 15). The present investigation performed in mice confirms and extends this concept. We showed that the iNKT cells that are required by tolerogenic APCs for the generation of Treg cells are CD4+ and must produce IL-4 and IL-10, but not IFN-{gamma} (Figs. 7 and 8). This strongly suggests that it is the CD4+ iNKT cell subset, and not the double-negative subset, that is required for oral tolerance induction in the model reported here.

We have demonstrated that iNKT cells are required not only during the induction of tolerance by oral administration of nickel, which occurs in the absence of costimulation, but also during the infectious spread of tolerance which is activated after immunization with NiCl2/H2O2. Although this immunization induces a specific DTH reaction in otherwise untreated mice (26), in the presence of tolerogenic donor APCs it apparently leads to the production by iNKT cells of IL-4 and IL-10 which in turn prevents the DTH response. This polarized cytokine response by the iNKT cells might be explained by the following two observations: 1) the donor APCs show a strongly reduced CD40 expression (21); and 2) it is these tolerogenic donor APCs (majoritively B cells), and not the APCs of the recipient, that are needed to activate the iNKT cells (Fig. 6). Due to their reduced CD40 expression, the tolerogenic donor APCs can only activate the iNKT cells via a CD40-independent pathway, which consequently leads to a Th2-cytokine production by the iNKT cells (48) and, hence, the prevention of DTH responses (49).

After the transfer of APCs from orally tolerized donors, the infectious spread of tolerance to T cells in the recipients requires immunization of the recipient with NiCl2/H2O2 (25). The reason why it takes immunization not only with NiCl2 but also with adjuvant, i.e., H2O2, for the tolerance spread remains yet unanswered. Several mechanisms can be considered to play a role here, in particular cell migration to and from the draining lymph node(s). In the first 3 days after the immunization, there is an up-regulation of CD80 and CD86 on the DCs of the draining lymph nodes (25), and possibly the nickel-reactive host T cells migrate there and undergo an initial activation before they are silenced. Whether or not the tolerogenic donor APCs and the iNKT cells interact with each other in the draining lymph node, the spleen, or both is unsolved. In any event, following our experimental protocol for the study of infectious nickel tolerance (25), the infected T cells of the first recipients were obtained from the spleens of the recipients.

In conclusion, although in the model studied here the Ag is administered orally whereas in ACAID it is injected intraocularly, there are a number of striking parallels between both models. The infectious tolerance cascade initiated by the transfer of only a few tolerogenic APCs in our model of oral nickel tolerance strongly resembles the cellular and molecular cascade underlying ACAID. In fact, ACAID might be considered an intraindividual infectious tolerance process. In both models, there is a requirement for CD1d-restricted CD4+ iNKT cells that produce IL-10 (in our model also IL-4) to translate and amplify the tolerogenic signals emitted by donor APCs to naive T cells and to render these cells suppressive. Extending the knowledge obtained from the ACAID model, we further demonstrated that once functional Treg cells were induced by oral nickel administration, they exerted their suppressive function independently of iNKT cells. Thus, iNKT cells serve to build up, but not to exert the specific suppressive mechanisms by Treg cells.


   Acknowledgments
 
We thank Dr. Günter Warncke and his team for management of the Umweltmedizinische Forschung animal house and breeding of the gene-defective mice used in the experiments, Birgit Steil for help in preparing the manuscript, and our colleagues at Umweltmedizinische Forschung as well as Marty Wulferink (Utrecht), Georg Kraal (Amsterdam), and Kate Heim (NiPERA) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by Grant SFB 503, "Molecular and cellular mediators of exogenous noxae," from Deutsche Forschungsgemeinschaft, Bonn, and by NiPERA, Research Triangle Park, NC. Back

2 Address correspondence and reprint requests to Dr. Ernst Gleichmann, Institut fuer Umweltmedizinische Forschung, Heinrich Heine University, Duesseldorf, Auf’m Hennekamp 50, D-40225 Duesseldorf, Germany. E-mail address: ernst.gleichmann{at}uni-duesseldorf.de Back

3 Abbreviations used in this paper: ACAID, anterior chamber-associated immune deviation; DTH, delayed-type hypersensitivity; i.d., intradermal; iNKT cells, invariant NKT cells; Treg cells, regulatory T cells; WT, wild type; DC, dendritic cell. Back

Received for publication March 16, 2004. Accepted for publication May 14, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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