Analysis of the mechanism(s) of immunological tolerance to a physiological soluble antigen in transgenic mice

The mammalian immune system recognizes and responds to a vast array of foreign molecules, and its diversity is generated by V-(D)-J gene recombination during ontogeny. The central feature of the immune system is self-nonself discrimination, which results in immunological reactivity or tolerance (specific immunological hyporesponsiveness to a previously encountered antigen-Ag). Breakdown of self-tolerance may result in autoimmunity to the host's own self-Ags or tissues. Recent studies have demonstrated that both central (thymus-dependent) and peripheral (extra-thymic) tolerance can operate in an Ag-specific manner by at least two major mechanisms, namely, clonal deletion (physical elimination of autoreactive lymphocytes) and clonal anergy (functional inactivation of autoreactive lymphocytes). A third mechanism, active suppression (functional inhibition of autoreactive lymphocytes), may also be of relevance, particularly for peripheral tolerance. We previously characterized the T-cell receptor repertoire specific for different insulins in Balb/c mice and identified a minimal immunogenic peptide (A chain: 1-13). These results prompted us to establish a transgenic (Tg) mouse model to study the mechanism(s) of tolerance to a soluble self-Ag under physiological conditions in vivo. These mice express the gene for a foreign Ag (beef insulin, BI, mutated from human insulin genomic DNA) under the regulatory control of its own promoter so that the Tg product is expressed in pancreatic $\beta$-islet cells. In this Tg mouse model, the functional expression of BI (range: 10$\sp{-10}$-10$\sp{-11}M$) is regulated by the host's glucose/insulin homeostasis, and is associated with a differential activation of BI-specific Th1/Th2 cells in vivo. These Tg mice are hyporesponsive to BI immunization at the level of both humoral and cell-mediated immune responses, mediated by Ag-specific T cells. Despite the level of no detectable thymic expression of the Tg products, BI-specific hyporesponsiveness may still occur in mature thymocytes of the Tg mice, suggesting the operation of a thymic selection process to a peripheral soluble Ag. Exogenous IL-2 can restore responses in peripheral T cells in vitro, suggesting also the involvement of an "anergy-inducing" mechanism. Adoptive transfer of some BI-specific CD4$\sp+$ Th2 cells from Tg mice into normal syngeneic Balb/c mice induced Ag-specific hyporesponsiveness (as determined by BI-specific ELISA). This in turn suggests an active suppression mechanism may be involved in the maintenance of peripheral self-tolerance. Using a transwell coculture system, it seems that the cytokine TGF-$\beta$, not IL-4 or IL-10, may be involved in suppression (but not killing or apoptosis) of Ag-specific Th1 cells. This active suppression is exerted in a bystander (Ag-specific and effector-nonspecific) fashion in vivo, and is associated with self-tolerance. The work presented in this thesis significantly extends the earlier findings in other soluble-Ag Tg model systems in which one dominant mechanism has been reported to explain the tolerance observed. Our results suggest that, at least for soluble Ags expressed peripherally: (1) self-tolerance can be accomplished both intra- and extra-thymically, and (2) there are multiple levels of regulation for self-tolerance in vivo, operating in an Ag-specific fashion, including thymic selection, peripheral anergy and active suppression. These results provide clear and important information which contribute to our understanding of the basic mechanism(s) involved in self-nonself discrimination in biological tolerance. Application of this information should improve our understanding and treatment of various diseases associated with dysregulation of immune responses, including autoimmunity, transplantation and perhaps malignancy.