Generation of a mouse model of extrathymically-derived regulatory T cells in type 1 diabetes

Abstract

Abstract Regulatory T cells (Tregs) are an immunosuppressive population that were initially identified as uniformly and stably expressing the transcription factor Foxp3. Tregs have now been further distinguished based on whether they developed in the thymus (thymically-derived Tregs, or tTregs) or in the periphery (peripherally-derived Tregs, or pTregs). Previous studies have shown that deletion of CNS1, a conserved non-coding DNA response element in the Foxp3 locus, leads to selective impairment of pTreg generation without disrupting tTreg generation in the B6 background. However, the role of pTregs versus tTregs in autoimmunity is unclear. Using CRISPR-Cas9, we removed the CNS1 region in the NOD mouse model of spontaneous type 1 diabetes. Based on in vitro Treg generation assays, removal of the CNS1 region impairs Foxp3 induction in naïve NOD CD4+ T cells. However, it did not lead to statistically significant differences in the percent of Tregs found in various tissues, including the spleen, thymus, gut, lymph nodes, and pancreatic islets of non-diabetic mice aged 8–13 weeks. Single-cell RNA seq comparing a 12-week old NOD mouse to a CNS1 KO NOD mouse also did not reveal strong differences in gene expression in the pancreatic islet, gut, or pancreatic lymph node, although flow cytometry analysis does reveal a small but significant decrease in RORyt+ Tregs and an increase in Helios+ Tregs in the gut of CNS1 KO NOD mice. CNS1 KO NOD mice are also being compared to normal NOD mice for type 1 diabetes onset and incidence. These preliminary data suggest that extrathymically-derived Tregs do not play a large role in this mouse model of type 1 diabetes.