Tolerance to immunologically privileged antigens that reside behind blood-tissue barriers, such as the eye and the brain, is still not understood completely. Studies in “shiverer” mice that lack myelin basic protein revealed that endogenous myelin basic protein expression is tolerogenic and alters the T cell repertoire selection (29
). The present study was undertaken to directly address the question whether tolerance to antigens residing in the retina of the eye can be demonstrated in mice that are susceptible to retinal autoimmunity. Previous data pointed to an association between presence of a particular retinal antigen in the thymus and resistance to EAU induced by immunization with that antigen (11
). However, the questions regarding uveitogenic antigens whose thymic expression is below the sensitivity of conventional assays could not be addressed until susceptible mice deficient for the target antigen became available.
It was first necessary to ascertain that the IRBP KO mice, which retain part of the IRBP gene, indeed fail to express antigenic protein transcribed from this sequence. This appeared to be the case, both by lack of immunoreactive material in the retina and by the resistance of these mice to an aggressive regimen of immunization with IRBP. Additionally, the immunological responses to IRBP of IRBP KO mice were elevated across the board, implying lack of systemic tolerance to this molecule. Thus, EAU susceptible WT mice displayed a detectable level of tolerance not present in the KO.
We then decided to examine the thymus of B10.RIII WT and KO mice for IRBP expression. PCR analysis of thymic extracts, under the conditions used by Egwuagu et al. (11
) to detect IRBP in resistant but not susceptible mouse strains failed to detect an IRBP signal, confirming what had been published. However, looking at individual cells by immunohistochemistry and microdissection, positive IRBP signals at the protein and the mRNA levels were detected in scattered cells in the medulla and cortico–medullary junction of WT thymus that were absent in KO thymus. Although we did not identify the cell type expressing IRBP in the thymus, it is likely that those were thymic medullary epithelial cells, as reported by others (35
Thymus transplantation between WT and KO mice provided direct evidence that this level of thymic expression is functionally significant in terms of setting the threshold of responsiveness to IRBP in general and the threshold of susceptibility to EAU in particular. Importantly, this included control of the response to the disease-relevant peptide 161–180, which is the single most dominant uveitogenic IRBP epitope for WT B10.RIII mice and accounts for much of the uveitogenicity of whole IRBP in that strain (10
). The frequency of p161–180-specific cells in WT mice and their apparent avidity was markedly reduced compared with the KO. It appears, therefore, that the 161–180-specific response that drives IRBP-induced EAU in WT mice represents a residuum of the original T cell repertoire generated to this epitope. The IRBP KO thymus might also permit retention of pathogenic repertoires to other IRBP epitope(s) eliminated in the WT, such as peptide 121–140.
Thymic control of T cell responses in the periphery involves not only the direct elimination of pathogenic cells but also the release of regulatory cells that control tissue-specific autoimmunity (for review see 32). It is thought that the relevant tissue antigen must be expressed in the thymus to permit generation of specific CD4+
regulatory T cells (33
). Enhanced responses to IRBP in mice bearing an IRBP KO thymus might therefore be attributable not only to culling of IRBP-reactive precursor-effector cells but also to lack of generation of CD25+
regulatory T cells specific to IRBP. It is important to point out in this context that although the protective role of CD25+
regulatory cells is well established in spontaneous autoimmunity models, it has been more difficult to demonstrate in induced models of autoimmunity and has not thus far been demonstrated in EAU. Furthermore, the eye elicits its own, highly specialized, immunoregulatory circuits that could make thymically derived CD25+
regulatory cells redundant (5
). Our finding that immunocompetent WT mice, deprived of thymus-dependent CD25+
regulatory cells, develop enhanced EAU scores, strongly supports a contributing role for these cells in setting the threshold of EAU susceptibility.
A role for peripheral mechanisms in self-tolerance to uveitogenic retinal antigens is still in question. Expression of retina-specific proteins is largely restricted to the eye, where blood-tissue barriers prevent free traffic of lymphocytes. Thus, peripheral tolerance by continuous exposure of potentially autoreactive T cells to tissue antigens in a noninflammatory milieu is unlikely to operate efficiently for retinal antigens. Indeed, we and others have demonstrated that responses to retina-specific antigens are dramatically reduced if their immune-privileged status is revoked by expressing them extraocularly (8
). It is still unresolved whether the ACAID phenomenon, which is well studied with relation to injected proteins (5
), is elicited by native retinal antigens residing in the intact eye. Gregerson et al. have proposed this to be the case based on reduced responsiveness to β-galactosidase in mice expressing this protein as a neo-antigen on a retina-specific promoter (39
), but they have not performed thymus transplants or CD25+
cell depletion to rigorously exclude central tolerance. Our results in mice immunoablated by irradiation clearly showed that WT mice given a KO thymus (whose only source of IRBP is the periphery), develop significant tolerance to IRBP compared with KO mice given a KO thymus (compare WTKO
mice in , a–h). However, although this demonstrates that peripheral tolerance can be induced in mice that have no thymic source of IRBP, it does not prove that the intact eye elicits such tolerance, since we cannot exclude the possibility that changes in the blood-retinal barrier resulting from irradiation have altered the accessibility of the ocular compartment. The question of peripheral tolerance by the intact eye is being addressed in a separate study.
Our current data complement and extend recent studies on thymic tolerance to proteolipid protein (PLP), one of the antigens driving disease in the experimental autoimmune encephalomyelitis model. Seminal studies by Klein et al. (40
) and by Anderson et al. (41
) revealed predominant expression in the thymus of DM20, a shortened splice variant of PLP message that lacks the region encoding the 139–151 epitope, instead of the full-length molecule that is expressed in the brain. This thwarts deletion of 139–151-specific T cells and explains the susceptibility of SJL/J mice to experimental autoimmune encephalomyelitis induced by this epitope but not by other PLP epitopes that are represented in the thymus. Unlike PLP, in the case of IRBP there is no evidence, either by PCR or by Western blotting, that the molecule expressed in the thymus represents a different splice variant than the one expressed in the eye (11
). Therefore, despite presence of full-length IRBP demonstrable at the protein level, elimination of pathogenic specificities is inadequate, although at the same time apparently permitting positive selection of CD25+
regulatory cells. These results raise a concept that the balance of autoimmune effectors versus regulators generated by the thymus may represent a sliding scale that is influenced by quantitative aspects of thymic autoantigen expression.
In summary, the present study for the first time provides direct evidence that uveitis-susceptible WT mice express low levels of their target antigen in the thymus. This level of expression, although undetectable by conventional assays, is immunologically relevant and serves to raise the threshold of immunological responsiveness and of susceptibility to disease. The mechanism appears to involve culling of precursor effector cells and generation of CD4+CD25+ regulatory cells by the thymus. These data provide new insights into regulation of responsiveness and autoimmune susceptibility to immunologically privileged self-antigens.