We have demonstrated that the loss of expression of a single self-antigen exclusively within the thymus can induce a spontaneous organ-specific autoimmune attack, even in the presence of functional aire. This result suggests that there are individual TSAs whose thymic expression is crucial in preventing autoimmune disease. Previous studies have suggested the importance of thymic tolerance and thymic TSA expression as a component in disease susceptibility (23
), but to our knowledge no previous study has demonstrated that the loss of a single TSA in the thymus is sufficient to induce organ- specific autoimmunity in a host with a polyclonal T cell repertoire. Unlike our results described in this paper, recent experiments with proinsulin 2 indicated that deficiency of this gene within the thymic compartment was not sufficient to induce diabetes (27
). In the case of insulin, peripheral tolerance mechanisms were likely sufficient to prevent overt autoimmunity, because a defect in central tolerance could be detected through an increase in insulin-specific T cells. In contrast, our results have identified an antigen for which central tolerance is critically important. It will be interesting to determine whether or not the phenomenon described in this paper is restricted to the eye. It is possible that we may have obtained this result because we focused on a site that has been described as immunologically privileged (28
). Perhaps because the immune system has limited access to the eye, there may be limited opportunity to induce tolerance via peripheral mechanisms, and this could increase the importance of central tolerance mechanisms for this target organ.
The level of IRBP expression within the thymus in our experiments was remarkably low. Despite this low level of expression, it is clearly enough to help impose tolerance in our model system, most likely through mechanisms that involve deletion rather than the positive selection of regulatory cells; in previous experiments, cotransfers of equal amounts of wild-type and aire-deficient thymi or lymphocytes were not capable of suppressing retinal disease (9
). Furthermore, it has been shown that T reg cells capable of suppressing EAU can be generated even in the absence of IRBP (29
). It remains to be understood how even this low level of expression allows for sufficient presentation to the developing thymocyte repertoire. Interestingly, because we could induce uveitis with transfers of IRBP-deficient/aire-sufficient thymic stroma, this process appears to be exclusively dependent on IRBP's expression and not another possible activity of aire such as antigen presentation, as invoked in a recent study (9
We have also confirmed one mechanism by which Aire operates to maintain self-tolerance. One of the current models for the function of Aire in maintaining self-tolerance has been that it is involved in the regulation of TSA expression in the thymus and that the loss of this function leads to a defect in negative selection of autoreactive thymocytes (3
). Microarray analysis of mTEC expression in the presence or absence of aire has helped identify many potential targets for autoimmunity in the aire-deficient model (3
), but data have been lacking on the actual primary targets to date. Two recent studies identified autoantigens targeted in the salivary gland and exocrine pancreas (30
) in the aire-deficient model whose expression was not regulated by aire in the thymus. However, these studies did not exclude epitope spreading as an explanation for their results. In contrast, through the use of IRBP/aire double-deficient animals, we have strong evidence that IRBP is the primary antigen driving the uveitis in these animals and is not an autoantigen by virtue of a secondary process like epitope spreading. These data help confirm the link between aire-driven regulation of TSA expression in the thymus and the development of a spontaneous autoimmune disease in animals with a polyclonal T cell repertoire. Our findings have also identified and confirmed that a single antigen is the primary target in a spontaneous autoimmune disease, something that has been extremely difficult to demonstrate in other spontaneous infiltrative autoimmune diseases, perhaps with the exception of insulin as a target in the NOD mouse model of diabetes (32
Interestingly, IRBP was not a predictable eye antigen in our model using the current mTEC expression profiling data (). Upon surveying the existing mTEC expression profiling data, we discovered that IRBP was not present on the microarrays used in these studies. This result suggests that there are many potential TSAs expressed in mTECs in an aire-dependent fashion that may be immunologically relevant but are not observed using the existing microarray analyses. Why IRBP is such a dominant and potent antigen in this system remains unclear. Our work suggests that there are properties of IRBP yet to be identified that make it immunogenic, which could include such processes as differential splicing, posttranslational modification, or antigen trafficking within and perhaps out of the eye. Along these lines, we also cannot completely exclude that the protective effect against eye autoimmunity in aire-deficient/IRBP- deficient animals is caused by secondary effects of the IRBP knockout that make the eye less immunogenic. Further work on IRBP as an autoantigen should shed light on the uveitic process that occurs both in these mice and in IRBP-induced EAU. IRBP is a large protein with multiple potentially immunogenic epitopes. However, it is not clear how and where IRBP peptides are presented to autoreactive cells. If priming occurs in the draining lymph nodes, by what route do IRBP-specific T cells gain access to the retina? A more detailed understanding of IRBP and IRBP-specific T cell responses should help elucidate additional aspects of disease pathogenesis and progression.
It is also tempting to speculate that susceptibility to EAU depends on a variation in IRBP expression within the thymic compartment across strains. In the EAU model, there are clearly strains that are resistant, protective, and susceptible. In our colony, both the EAU-resistant C57BL/6 and EAU-protected BALB/c strains are susceptible to spontaneous uveitis when deficient for aire (unpublished data) (3
). This leaves open the possibility that when IRBP is removed from the thymus by aire deficiency, the strain dependency for EAU is removed; in fact, there are data to support strain differences in eye antigen expression in the thymus (33
Collectively, our results show that thymic tolerance can be crucial in preventing autoimmune disease and that even the loss of expression of a single TSA in the thymus can generate spontaneous autoimmunity. It will be interesting to identify other thymic TSAs that behave similarly to IRBP and also determine if this observation is applicable to autoimmunity in human subjects given the link to the APECED syndrome.