A tale of two transgenic models: The role of central and peripheral mechanisms of T cell tolerance to the acetylcholine receptor

In older individuals, the decline in immunocompetence is often accompanied by an increase in the levels of circulating autoantibodies. To address the effects of aging on B cell tolerance, this study assessed the immune response to the acetylcholine receptor (AChR), the autoantigen in Myasthenia gravis (MG). Using an induced murine model of MG in which C57Bl/6 mice are immunized with acetylcholine receptor isolated from the electroplax organs of Torpedo californica (TAChR), we were able to assess whether young and old TAChR immunized mice would make antibodies that would cross-react with the endogenous mouse AChRalpha antigen. After multiple immunizations with the inducing antigen, TAChR, antibody titers to the recombinant murine AChRalpha (mAChRalpha1-210) were significantly lower than to TAChR(alpha1-210) in both young and old animals suggesting B cell tolerance was maintained with age.;In MG, the anti-AChR antibody response is T cell dependent; therefore, to assess mechanisms of T cell tolerance to this muscle autoantigen, two transgenic mouse models (Tg1 and Tg3) in which the TAChRa chain is expressed as a self protein were employed. The TAChRa transgene was expressed 3.5-fold higher in muscle and approximately 2-fold higher in thymus of Tg3 mice compared to Tg1. These models were used to investigate the role of Aire (a&barbelow;utoi&barbelow;mmune r&barbelow;egulator), a transcription factor critically important for central tolerance to several other peripheral antigens, in T cell tolerance to AChR. Tg1 and Tg3 mice remained tolerant to p146-162 despite being deficient in Aire. This was likely due to the fact that the TAChRa transgene was not under Aire control in either Tg1 or Tg3 mice, which may be physiological since the endogenous mouse AChR was only shown to decrease 2-fold in Aire knockout mice.;Thymus transplants were then performed to assess whether thymic expression was required for the generation of tolerance to AChR, the first muscle autoantigen investigated. Surprisingly, in Tg1 mice, peripheral expression of TAChRalpha alone was sufficient to induce tolerance to the AChR, although thymic expression of the transgene could elicit tolerance in 3/8 transplant mice. On the other hand, tolerance was consistently observed in non-transgenic mice grafted with a Tg3 thymus. Thus, both central and peripheral mechanisms are likely to play a role in T cell tolerance to AChR.;To explore which mechanisms of peripheral tolerance were involved, CFSE-labeled TAChR specific T cells from a TCR transgenic were adoptively transferred into Tg1 and Tg3 transgenic and non-transgenic recipients. Following immunization, proliferation of the transferred T cells was apparent in both transgenic (Tg1 and Tg3) and non-transgenic hosts suggesting anergy and deletion were not likely mechanisms of tolerance. When Tg1 mice were bred to scurfy (Foxp3 sf) mice, progeny lacking T regulatory cells (Tregs) exhibited an increase in serum anti-TAChR antibodies, yet elevated antibody titers were not seen in the Foxp3wt mice or in mice lacking Aire function. Furthermore, inactivation of Tregs using an anti-CD25 antibody yielded a loss of T cell tolerance in both Tg1 and Tg3 mice. We also showed that MG could be induced in 46% (6/13) of Tg1 and Tg3 mice in which Tregs were functionally depleted compared to 8% (1/13) of mice treated with an isotype-matched control antibody. Thus, these studies support a predominant role for Tregs in tolerance to the AChR and suggest Tregs are likely involved in MG disease induction as well. Overall, the Tg1 and Tg3 models are important tools by which the relative contribution of both central and peripheral mechanisms of T cell tolerance to the AChR can be assessed with the goal of enhancing therapeutic strategies for patients with MG.