Thymic Development of β Cell Specific Regulatory T Cells in Neonatal NOD mice

Neonatal period is a critical window when central tolerance is established in an Autoimmune Regulator (Aire). However, not all self-antigens are expressed downstream of Aire and tolerance to some tissue restricted antigens might rely in part on peripheral dendritic cells (DCs). The relative contribution of thymically and peripherally derived antigens in the generation of neonatal β cell antigen specific CD4⁺ regulatory T cells (Tregs) is not well understood. We have recently shown ectopic expression of Insulin B_9-23 and the insulin mimetope R22E within antigen presenting cells early in thymocyte development can protect mice from developing Type 1 Diabetes (T1D) through central tolerance mechanisms. However, it remains to be determined whether peripherally derived neo-antigens such as post-translationally modified (PTM) peptides, may also be transported to the thymus to impact thymocyte development. Pathogenic T cells that are specific for neo-antigens uniquely expressed in the pancreas may escape thymic selection. Therefore, we examined the role of a known PTM peptide, the hybrid insulin/chromogranin A (2.5HIP) peptide, on neonatal development of β cell specific T cells and Tregs. We found that adoptively transferred DCs pulsed with ChgA peptides into neonatal and adult BDC2.5 TCR Tg and WT NOD mice readily migrate to the thymus and impact the development of ChgA specific T cells. Additionally, we found targeting thymic Langerin+ dendritic cells with anti-Langerin linked to ChgA peptides also altered T cell development. In both experimental approaches, we found a robust increase in the ratio and number of ChgA specific Tregs in the presence of 2.5HIP PTM while also observing an increase in negative selection. Co-administration of exogenous antibody/IL-2 complexes greatly enhanced the frequency and number of thymic Foxp3+ Tregs. Together our data suggests neonatal exposure to PTM peptides can efficiently drive thymic development of β cell specific Tregs and may alter T1D pathogenesis.