Data collected in this study show that transcripts of all the eight tested tissue antigens are expressed in detectable levels in the thymus of most individuals. This observation is in line, therefore, with the notion that essentially all peripheral tissue-specific antigens of the mammalian body are normally expressed in the thymus, thus initiating the mechanism of central tolerance whereby T lymphocytes with high affinity toward these self-antigens are eliminated (2
). Our data are also in accord with the recent finding by Gotter et al.
), that human medullary thymic cells over-express >400 genes, including many genes of tissue-specific antigens.
Remarkable variations were noted, however, in the present study among the tested thymic samples in their level of expression of the different transcripts ( and ). This variability resembles the one observed among different inbred strains of mice and rats, when tested for thymic expression of immunopathogenic antigens of the retina (4
). As reported in these animal studies, an inverse correlation was found between thymic expression of immunopathogenic antigens and the susceptibility of the rodent strain to experimental autoimmune diseases induced by the tested antigen (4
). It is conceivable, therefore, that similar to the rodent system, the level of thymic expression of potentially immunopathogenic antigens plays a role in determining the susceptibility of humans to the development of autoimmunity against the antigens.
The variability in thymic expression of the retinal genes among different donors, as detected by the RT–PCR method, was confirmed by repeated experiments, as depicted in . It is noteworthy that the gene expression profile of individual donors was homogeneously distributed throughout the different areas of the thymus, as shown in .
Our results with the quantitative real-time PCR analysis underscored how minuscule is the amount of S-Ag in the human thymus, as compared with the level of this molecule in the retina (). This observation with human tissues is in line with data collected with experimental animals, with both native and neo-self ocular antigens (29
). It is notable, therefore, that trace amounts of ocular-specific antigens expressed in the animal thymi were found to efficiently eliminate large populations of thymocytes with high avidity to these ocular antigens (1
). It is assumed that similar processes of specific lymphocyte elimination take place in the human thymus.
The expression of a wide range of tissue-specific antigens in human thymi was investigated by Sospedra et al.
). One of the molecules included in that study was retinal S-Ag. Strong RT–PCR responses for the S-Ag transcript were found in only three of the 12 tested thymic samples, from donors of 2, 5 or 13 months of age, but no activity was detected in thymi from nine other donors, aged from 8 days to 13 years. This ‘clustering’ of responders at a specific age range is contradicted by our data, showing that transcripts of S-Ag and other retinal antigens were found in thymi collected at all tested ages. It is possible that factors related to the RT–PCR technique are responsible, at least in part, for this observation by Sospedra et al.
). In a more recent paper, Gotter et al.
) detected transcripts of S-Ag and IRBP in thymic medullary cells of all five tested donors. In addition, these authors detected in the medullary cells transcripts of several tumor-associated antigens (28
). It is of note that our observation with melanocyte transcripts () supports the conclusion of Gotter et al.
) concerning the controversial issue of thymic expression of tumor antigens.
The thymic selection process is incomplete and T cells with low avidity toward the self-antigen do escape elimination (26
). Consequently, lymphocytes that recognize various tissue antigens, including retinal proteins, are found in healthy individuals (32
). It is conceivable that the avidity of these T-cell escapees is determined by the level of the corresponding tissue antigen in the thymus. This issue is of particular interest in view of the finding that the avidity toward myelin antigens of T cells from patients with multiple sclerosis is significantly higher than that of cells from healthy controls (34
). Likewise, the avidity toward the target melanocyte antigens determines the therapeutic capacity of T cells used in immunotherapy of melanoma activity and, therefore, the level of thymic expression of these antigens is likely to have a pivotal effect on the success of cell-mediated immunotherapy of melanoma (11
The RT–PCR data collected in this study are also of interest with regard to cellular immune responses of humans toward retinal antigens. Lymphocyte responses have been tested against three of the four retinal antigens used here, i.e. S-Ag, recoverin and IRBP. A majority of the tested human subjects was found to respond to S-Ag (32
), whereas most subjects were found to show no response to recoverin (35
) and very low or no response to IRBP (33
). The prevalence of the response to S-Ag in healthy donors resembles that to MBP (34
) or PLP (38
) and it is assumed that cells that respond to these tissue-specific antigens are T lymphocytes that escaped negative selection. In view of the general similarity between thymic expression of S-Ag transcript and of IRBP or recoverin transcripts (), the difference between the responses to these antigens suggests that factors other than thymic expression of the retinal antigen are also involved in determining the responsiveness of human lymphocytes to tissue-specific antigens.
All three tested retinal antigens, S-Ag, IRBP and RPE65, were detected in human thymic sections by immunostaining, using specific antibodies against these proteins. The antigens were detected in rare cells located mostly in the thymic medulla, thought to be the site where self-antigens are expressed and the negative selection occurs (26
). These observations resemble closely those made with other tissue antigens, such as those of the pancreatic islets (25
In summary, our data show that thymic expression is a common feature of uveitogenic retinal and melanocyte antigens in humans and further suggest that thymic expression is common to all human tissue-specific antigens. Remarkable variations were noted, however, among individual thymi, suggesting that the level of thymic expression of any tissue antigen could affect the presence and avidity of T lymphocytes specific to these antigens. Consequently, the thymic expression of uveitogenic antigens could affect the susceptibility of the individual to pathogenic autoimmunity against the tissue molecule.