The Role of Recirculating Thymic T regulatory cells in Central Tolerance and Immune Homeostasis 2261236

Abstract

Abstract Introduction AIRE+ medullary thymic epithelial cells (mTECs) play a critical role in establishing immune tolerance. These cells peak 2-3 weeks after birth and then steadily decline as mice get older. The mechanism by which AIRE+ mTECs decline remains unclear. We observed that accumulation of recirculating thymic T regulatory cells (RT-Tregs) coincides with a decline in AIRE+ mTECs. Using scRNA-seq, we found that RT-Tregs may have cytotoxic potential. We studied the effect of RT-Tregs on AIRE+ mTEC abundance by selectively depleting RT-Tregs. Methods Thymi samples of RagGFP x Foxp3RFP mouse at age 1, 2, 3, 4, 8, and 12 weeks (n = 4) were analyzed using flow cytometry. Single-cell RNA-seq analysis was performed on CD73+ thymocytes in 8-9-week-old mice. Ultrasound-guided intrathymic injection of 0.05 μg diphtheria toxin (DT) or PBS was performed on 5-week-old Foxp3-DTR mice (n = 8) every other day for 1.5-4.5 weeks and AIRE+ mTECs were quantified using flow cytometry. Results scRNA-seq analysis revealed different clusters within RT-Tregs, such as effector Treg subset including GzmB+ cells. RT-Tregs accumulate in the thymus after birth and reach up to 26.1% of the Treg compartment by 4 weeks of age, and 73.9% by 12 weeks of age. Upon intrathymic DT injection, RT-Treg population was reduced significantly (>50%) but de novo and splenic Tregs were preserved. RT-Treg depletion resulted in a significant increase (p = 0.008) in the number of AIRE+ mTECs. Conclusion We show that RT-Tregs are a diverse subset that includes effector-like cells. When RT-Treg number increases, AIRE+ mTEC decreases, and vice versa. Using previously published datasets, we observed that TCR diversity of conventional T cells (Tconv) in lymphoid organs of AIRE-/- mice was significantly increased relative to WT mice. We hypothesize that early in life AIRE+ mTECs promote self-tolerance. Once the peripheral tolerance has been established, RT-Tregs eliminate AIRE+ mTECs, resulting in a more diverse TCR repertoire that is more pathogen-reactive. Funding Source n/a Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)