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

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-PtPrc^a Pep3^b/BoyJ), IL4^−/− (C57BL/6-IL-4^tm1Nnt), IL-10^−/− (C57BL/6-IL-10^tm1Cgn), and IFN-γ^−/− (C57BL/6-Infg^tm1Ts) females were purchased from The Jackson Laboratory (Bar Harbor, ME). Jα18^−/− 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-CD3ε (clone 145-2C11); PE- or PerCP-labeled anti-CD4 (clone RM4-5); PE-labeled anti-CD8β.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-A^b (clone AF6-120.1); PE- or PerCP-labeled anti-NK1.1 (clone PK136); and FITC-labeled anti-TCRβ-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 × 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-II^high 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-II^high 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% TCRβ⁺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.

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). Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− mice that were immunized with NiCl₂ in the absence of H₂O₂ only showed a background response (Fig. 1⇓, bar 5). The tolerance induced in Jα18^−/− 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, Jα18^−/− mice fail to develop nickel tolerance after a 4-wk course of 10 mM NiCl₂ in the drinking water. WT and Jα18^−/− 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, Jα18^−/− 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, Jα18^−/− mice, which were orally either treated with NiCl₂ or not, were immunized and rechallenged with DNFB (bars 6 and 7); untreated Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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.

Jα18^−/− mice possess tolerogenic APCs after the oral administration of nickel, but lack Treg cells. Prospective WT and Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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.

Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− recipients. However, 10⁴ WT donor Treg cells depleted of NK1.1⁺ cells were still able to transfer the tolerance to Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− (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 Jα18^−/− donors were eliminated from those of the first set of recipients before the second transfer. Because the Jα18^−/− 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. 5⇓A). 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. 5⇓A, 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 Jα18^−/− donors could infectiously spread the tolerance to T cells of naive recipient mice. Thus, the APCs of orally treated Jα18^−/− mice were capable of inducing Treg cells, notwithstanding the fact that the orally treated Jα18^−/− 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⁺ Jα18^−/− (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 Jα18^−/− (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 Jα18^−/− 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. 5⇑B, bars 1 and 2), and Ly5.2⁺ Jα18^−/− mice as the first recipients (Fig. 5⇑B, 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. 5⇑B, bar 4). This is evident from the fact that adoptive transfer of T cells from Jα18^−/− 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 Jα18^−/− mice (Fig. 1⇑). However, differences did emerge when APCs from these orally treated Jα18^−/− and CD1^−/− donors were adoptively transferred into WT recipients (Fig. 6⇓). Whereas the APCs of orally treated Jα18^−/− 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, Jα18^−/−, 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 Jα18^−/− 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 Jα18^−/− recipients when the latter were reconstituted with iNKT cells. As a source of iNKT cells, splenocytes (10⁷) from naive WT donors were used (Fig. 7⇓A). As expected, the transfer of naive spleen cells alone failed to tolerize the Jα18^−/− recipients, as did the transfer of 10⁵ tolerogenic APCs from orally tolerized WT donors (Fig. 7⇓A, 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 Jα18^−/− recipients (Fig. 7⇓A, bar 4). The results shown in Fig. 7⇓B demonstrate that the iNKT cells within the naive WT spleens used to reconstitute the Jα18^−/− recipients were, indeed, the missing cell population. To be more exact, tolerogenic WT APCs failed to induce tolerance in Jα18^−/− recipients when they were cotransferred with naive spleen cells from Jα18^−/− donors (Fig. 7⇓B, bar 3) or with either spleen cells from naive WT donors after the depletion of NK1.1⁺ cells or CD4⁺ cells (Fig. 7⇓B, bars 4 and 5). Therefore, the data suggest that reconstitution with CD4⁺ iNKT cells enables Jα18^−/− 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 Jα18^−/− recipients. Prospective WT donor mice were orally tolerized or were left untreated, and naive Jα18^−/− mice served as recipients. A, Jα18^−/− 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 Jα18^−/− 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 Jα18^−/− 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

Jα18^−/− 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-γ^−/− donors into Jα18^−/− recipients (Fig. 8⇓). The percentage of T cells (both CD3⁺CD4⁺ and CD3⁺CD8⁺), NKT cells (NK1.1⁺TCRβ⁺), B cells (B220⁺MHCII⁺), and DCs (CD11c⁺MHCII^high) 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-γ^−/− 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-γ-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-γ^−/− donors were isolated, mixed, and injected i.v. into naive Jα18^−/− 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.