In the present study we demonstrate that iNKT cells can have a role in EAU regulation. Their role appears to be not in setting the threshold of susceptibility to EAU, as do the natural CD4+CD25+ regulatory cells whose function in deterring development of ocular autoimmunity we have characterized in recent studies (30
). Rather, they can inhibit developing disease following a pharmacological enhancement of their activity at or around the time of priming. In chronic autoimmunity, priming of new effector T cells is believed to be occurring on a continuous basis. Since endogenous ligands for iNKT cells exist in the body and can trigger iNKT activity (4
), it is conceivable that iNKT cells can participate in modulating the course of ocular autoimmune disease. Thus, there appears to be a “division of labor” between the natural CD4+CD25+ regulatory T cells and iNKT cells, with the former setting the threshold of susceptibility, and the latter possibly regulating the autoimmune response after that threshold has been passed.
The groups of Streilein and Stein-Streilein demonstrated that iNKT cells have a central role in ACAID, a prototypic regulatory phenomenon elicited by injection of Ag into the anterior chamber of the eye and its transport by eye-derived APC to the spleen (10
). iNKT cells are recruited into the spleen via a mechanism involving MIP-2 and participate in priming the adaptive T regulatory cells typically associated with ACAID. Although prior elicitation of ACAID to IRBP can inhibit a subsequent episode of EAU (32
), it is unlikely that the protection from EAU by iNKT that we observe here bears a relationship to their role in ACAID. In ACAID the eliciting Ag originates from the eye and the eye has to be perturbed (injected with Ag) in order for this phenomenon to be observed, and iNKT activation, if any, occurs without additional manipulation. In contrast, in our study, pharmacological activation of iNKT cells is needed and is applied when the eye is still intact. Thus, it is conceivable that iNKT cells may regulate ocular immune responses at more than one level.
Studies in the models of experimental arthritis, NOD diabetes and EAE (reviewed in 22
) had indicated that activation of iNKT by OCH is more effective than by α–GalCer, which was attributed to its induction of IL-4 and Th2 skewing. We were therefore surprised to find that OCH was not more effective than α–GalCer in protecting from EAU, and that the most efficient protection followed administration of α-C-Gal-Cer, which induces an IFN-γ dominated iNKT cytokine response. Thus, effectiveness of protection paralleled the innate IFN-γ inducing ability of the invariant TCR ligand. The protection was accompanied by reduction in the IRBP-specific adaptive Th1 and Th17 pathogenic effector responses, as judged by production of their respective hallmark cytokines IFN-γ and IL-17 to in vitro
recall with IRBP. The functional role of IFN-γ in the protective and regulatory effects of iNKT are strongly supported by direct evidence showing that neutralization of innate IFN-γ reversed the protective effect of α-C-Gal-Cer and restored the subsequent proinflammatory cytokine production of the adaptive response. This is not to say that the mechanism of protection is the same for all the three analogs. Our data do not negate the possibility that protection from EAU by OCH and by α–GalCer could involve IL-4, as was previously demonstrated in several other autoimmune disease models (22
Our data are in line with some previous reports, which revealed that protection from autoimmune disease by iNKT may not always involve IL-4 and Th2 skewing. Studies by Lehuen and her colleagues demonstrated that even in the absence of IL-4 iNKT cells can control EAE (33
) and experimental type 1 diabetes (T1D) (34
). This was associated with a decrease in Th1-associated pathogenic autoimmune responses without inducing Th2 responses, and was due at least in part to induction of anergy in the autoreactive T cells (35
). Such a mechanism could also be involved in the prevention of EAU observed here. Although these studies did not directly implicate IFN-γ in these effects, participation of IFN-γ (rather than IL-4) in protection from EAE was suggested by Furlan et al. (27
It should be noted that high systemic levels of IFN-γ early or late in the disease can be protective, but likely by different mechanisms. Initial production of IFN-γ would be mostly from NKT and NK cells, whereas later in disease Ag specific Th1 cells are a major source of IFN-γ. We previously showed that early upregulation of IFN-γ by injections of IL-12 inhibits development of EAU and associated immunological responses by aborting priming, through a process that involves induction of nitric oxide and apoptosis (29
). The innate IFN-γ produced by α-C-Gal-Cer-triggered NKT cells may well work in a similar fashion. On the other hand, protective effects of IFN-γ later in the disease appear to be due to its role in elimination of spent effector cells by activation-induced cell death (36
). Thus, neutralization of systemic IFN-γ at that stage also enhances disease (19
), although at that point it is not possible to distinguish between effects of IFN-γ produced by Ag specific T cells and iNKT cells.
In summary, we have demonstrated that iNKT cells can actively participate in regulating the autoimmune response to immunologically privileged retinal Ags. This apparently occurs at a different level than their role in induction of ACAID. The mechanism involves the induction of innate IFN-γ production through ligation of the invariant TCR and results in inhibited development of adaptive Th1 and Th17 responses that represent pathogenic effector mechanisms in uveitis.