AGING-INDUCED INCREASED SUSCEPTIBILITY TO AUTOIMMUNITY IS DUE TO COMPROMISED NEGATIVE SELECTION IN THE THYMUS RATHER THAN DEFECTS IN REGULATORY T CELLS

Purpose: Immunotolerance generates protection against autoimmunity by deleting self reactive T cells in the thymus through negative selection as well as the generation of natural regulatory T cells (nTregs) that will help suppress autoimmunity in the periphery. Natural aging is associated with a progressive loss of FoxN1 and thymic atrophy, and is also believed to be associated with increased autoimmunity and an increase in suppressive FoxP3+ regulatory T cells (Tregs). Deletion of self-reactive T cells requires self-antigen presentation by medullary thymic epithelial cells (mTECs). We set out to determine if thymic aging, characterized by the progressive loss of FoxN1 and mTEC disruption, alters immunotolerance by influencing negative selection or impacting the generation of suppressive Treg cells. Methods: For thymus population experiments, we induce thymic atrophy in our FoxN1 conditional knockout mice using tamoxifen injections. We then determine expression of CD4+, CD8+, and CD4+8+ double positive, and CD25+FoxP3+ regulatory thymocytes using flow cytometry. The peripheral Treg data is assessed by adoptive transfer of total spleenocytes from young and aged wild type mice into young Rag2-/- mice. Cell populations are determined using flow cytometry. Results: We found that the loss of FoxN1 induced thymic atrophy is associated with an impairment of negative selection, where CD4+ and CD8+ are increased in the thymus while CD4+CD8+ double positive thymocytes are decreased. This indicates that the age atrophied thymus is not able to delete additional single positive thymocytes, and these may be self-reactive thymocytes. Additionally, we found that nTregs in the aged and atrophied thymus are increased in proportion and their suppressor function remains intact. Furthermore, we found that when we transfer aged spleenocytes, in which there are increased Treg and decreased pro-apoptotic Bim protein, and young spleenocytes separately into young Rag2 knockout mice, that the young periphery is able to restore both Bim levels and Treg levels to that of the young mice. Conclusions: We conclude that loss of FoxN1 disrupts thymic mTEC structure and impairs negative selection, which may lead to an increase in self-reactive T cells. However, thymic atrophy does not compromise peripheral Tregs. The function and number of peripheral Tregs is dependent on the micro-environment in which they stay.