Abstract

Myelin reactive T cells are central in the development of the autoimmune response leading to central nervous system (CNS) destruction in Multiple Sclerosis (MS) and its animal model, Experimental Autoimmune Encephalomyelitis (EAE). The underlying cellular and molecular mechanisms, however, are not fully understood. In previous mouse studies, we showed that tolerance to the major component of the myelin sheath, myelin proteolipid protein (PLP), is crucially dependent on its expression in the thymus where central tolerance induction takes place. To analyze the phenotypic and functional changes taking place during the induction of tolerance in the thymus, we investigated the fate of PLP autoreactive CD4+ T cells in TCR-PLP11 transgenic mice, which express a transgenic TCR specific for the dominant PLP174-181 epitope in B6 mice, a EAE-resistant mouse strain of the H-2b haplotype. In previous work we found that a fraction of CD4+ T cells specific for this region appear to escape from tolerance induction. Our data showed that in TCR-PLP11 PLPWT mice, where PLP is transcribed in the thymus similar numbers of CD4+ thymocytes developed, compared to TCR-PLP11 PLPKO mice where PLP expression in the thymus is absent. This indicated that PLP174-181-specific thymocytes were not negatively selected. In the periphery, the PLP174-181-specific T cells displayed a naïve phenotype and therefore were not tolerized by clonal deletion or anergy induction. Potentially autoreactive CD4+ T cells were found in the spleen and lymph nodes of TCR-PLP11 mice but only became activated when stimulated in vitro. These cells were not spontaneously activated in vivo, indicating that PLP is not expressed/presented in the periphery. TCR-PLP11 mice do not develop any clinical or histological signs of EAE. Therefore, ignorance but not deletional tolerance is considered as main tolerance mechanism to avoid CD4+ T cell-mediated autoimmunity in our system. That means that naïve autoreactive CD4+ T cells ignore PLP antigens and recirculate in the periphery without causing damage. In contrast, immunization of TCR-PLP11 PLPWT mice with the PLP174-181 peptide in Complete Freund´s Adjuvant (CFA) reversed this state of immune ignorance as judged by the clinical manifestations of EAE in these mice. Furthermore, TCR-PLP11 PLPWT mice develop spontaneous EAE after being bred onto a RAGKO background, leading us to the speculation that, besides immunological ignorance, dominant mechanism of PLP tolerance are crucial for the prevention of CNS autoimmunity. Taken together, this study establish a novel model of immunological tolerance towards a self-antigen expressed in the central nervous system involving antigen ignorance of CD4+ T cells. Somatic recombination of TCR genes in thymocytes not only results in the production of useful TCR specificities, but also produces potentially autoreactive specificities. Autoreactive CD4+ T cells are censored by two mechanisms, the so-called clonal deletion and the deviation into regulatory T cells in the thymus both requiring the same stimulus, namely the interaction with self-peptide. In order to study the mechanisms of central and peripheral tolerance that operate to shape the CD4 T-cell repertoire, we have generated a TCR transgenic mouse that expresses the α- and β-chains of a PLP11-18-reactive TCR (TCR-PLP1) in the context of H-2b. Using this novel TCR-PLP1 transgenic mouse model, we investigated the modalities of central tolerance induction to a self-antigen expressed in the thymus at physiological levels. We found that Plp1-specific T cells undergo clonal deletion and Treg differentiation concomitantly upon encounter of the cognate self-antigen PLP. Medullary thymic epithelial cells (mTECs) express and present the endogenous antigen PLP and mediate tolerance in an autonomous manner, whereas thymic dendritic cells are dispensable for central tolerance induction to PLP. Although central tolerance induction to PLP is very potent, it is not complete as a proportion of autoreactive T cells also escape to the periphery. However despite the presence of potentially dangerous cells in the periphery, the mice do not develop autoimmunity indicating that additional tolerogenic mechanisms promote tolerance to PLP in the periphery. We could further show that autoreactive Plp1-specific T cells are deleted or become functionally inactivated (anergy) by recognition of self peptide on dendritic cells in the periphery. In a set of bone marrow transplantation experiments we found that PLP was expressed by radioresistant stromal cells and subsequently cross-presented by dendritic cells. Taken together our results indicate a complementing role of the thymus and the periphery for tolerance induction to PLP.