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 Jalpha18(-/-) mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those Jalpha18(-/-) 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 Jalpha18(-/-) 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, Jalpha18(-/-) 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.

Previously, we reported that tolerance to nickel, induced by oral administration of Ni2+ ions, can be adoptively transferred to naive mice with only 102 splenic T cells. Here we show that 102 T cell-depleted spleen cells (i.e., APCs) from orally tolerized donors can also transfer nickel tolerance. This cannot be explained by simple passive transfer of the tolerogen. The APCs from orally tolerized donors displayed a reduced allostimulatory capacity, a tolerogenic phenotype, and an increased expression of CD38 on B cells. In fact, it was B cells among the APCs that carried the thrust of tolerogenicity. Through serial adoptive transfers with Ly5.1+ donors and two successive sets of Ly5.2+ recipients, we demonstrated that nickel tolerance was infectiously spread from donor to host cells. After the transfer of either T cells or APCs from orally tolerized donors, the spread of tolerance to the opposite cell type of the recipients (i.e., APCs and T cells, respectively) required recipient immunization with NiCl2/H2O2. For the spread of tolerance from a given donor cell type, T cell or APC, to the homologous host cell type, the respective opposite cell type in the host was required as intermediate. We conclude that T suppressor cells and tolerogenic APCs induced by oral administration of nickel are part of a positive feedback loop that can enhance and maintain tolerance when activated by Ag associated with a danger signal. Under these conditions, APCs and T suppressor effector cells infectiously spread the tolerance to naive T cells and APCs, respectively.

Whereas oral nickel administration to C57BL/6 mice (Ni(high) mice) renders the animals tolerant to immunization with NiCl2 combined with H2O2 as adjuvant, as determined by ear-swelling assay, it fails to tolerize Jalpha18-/- mice, which lack invariant NKT (iNKT) cells. Our previous work also showed that Ni(high) splenic B cells can adoptively transfer the nickel tolerance to untreated (Ni(low)) recipients, but not to Jalpha18-/- recipients. In this study, we report that oral nickel administration increased the nickel content of splenic Ni(high) B cells and up-regulated their Fas expression while down-regulating expression of bcl-2 and Bcl-xL, thus giving rise to an Ag-carrying, apoptosis-prone B cell phenotype. Although oral nickel up-regulated Fas expression on B cells of both wild-type Ni(high) and Jalpha18-/- Ni(high) mice, only the former showed a reduced number of total B cells in spleen when compared with untreated, syngeneic mice, indicating that iNKT cells are involved in B cell homeostasis by eliciting apoptosis of effete B cells. Upon transfer of Ni(high) B cells, an infectious spread of nickel tolerance ensues, provided the recipients are immunized with NiCl2/H2O2. As a consequence of immunization, Fas ligand-positive (FasL+) iNKT cells appeared in the spleen and apparently elicited apoptosis of Ni(high) B cells. The apoptotic Ni(high) B cells were taken up by splenic dendritic cells, which thereby became tolerogenic for nickel-reactive Ni(low) T cells. In conclusion, FasL+ iNKT cells may act as ready-to-kill sentinels of innate immunity, but at the same time assist in tolerance induction by eliciting Fas/FasL-mediated apoptosis of effete, Ag-containing B cells.

The dose dependence of oral nickel tolerance was analyzed by comparing three different subsets of C57BL/6 mice: Ni(very low) mice were reared in a nickel-reduced environment, Ni(low) and Ni(high) mice were reared in a stainless steel-containing environment and the latter received oral NiCl(2) (10 mM). In spleen and feces, Ni(very low) mice exhibit significantly lower nickel concentrations than Ni(low) and Ni(high) mice. In contrast to Ni(very low) mice that can be sensitized with a single intradermal administration of NiCl(2) alone, Ni(low) mice can only be sensitized in the presence of an adjuvant and Ni(high) mice cannot be sensitized at all. This dose-dependent resistance to nickel sensitization (i.e. Ni(high) > Ni(low) > Ni(very low)) correlates with differences in the number and type of nickel-specific T regulatory (Treg) cells. Adoptive transfer studies into Ni(very low) recipients showed that Ni(very low) mice completely lack specific Treg cells whereas Ni(low) and Ni(high) mice harbor them, albeit their numbers and/or suppressive strength are much higher in Ni(high) than Ni(low) mice. The principal Treg subset in Ni(low) mice consists of CD4(+)CD25(+) cells, among which CD4(+)CD25(+)alpha(E)beta(7)(+) cells are the most effective. In Ni(high) mice, CD4(+)CD25(+) Treg cells co-exist with an ensemble of CD8(+) Treg and CD4(+)CD25(-) suppressor-inducer cells.