CD4 and CD8 double positive T cell progenitors in mediastinal adipose tissue and the aortic arch differentiate to CD4 T cells

Using 10× Genomics-based single-cell RNA sequencing on digested atherosclerotic mouse aortas, we discovered an unexpected immune cell population whose transcriptome was similar to thymic CD4+CD8+ cells (DPs). Flow cytometry showed DP presence in the aortic arch (AA) and mediastinal adipose tissue (MAT), but not in lymph nodes, spleen, blood or other tissues. DPs in MAT or AA did not change with atherosclerosis in Apoe−/− mice. Fluorescence microscopy of cleared whole mounts placed DPs in the AA adventitia and near microvessels in MAT. MAT and AA DPs were absent in Rag2−/− mice and in day3 and 21 thymectomized mice. Deep transcriptional profiling from sorted thymic, MAT, and AA DPs showed hundreds of differentially expressed genes. While thymic DPs were enriched for TCR signaling pathways, extra-thymic DPs were enriched for stress responses and cell cycle pathways. Pulse-chase experiments demonstrated a five-day turnover of MAT and AA DPs. Functionally, sorted MAT DPs developed successfully into conventional T cells (with a bias to CD4) in hanging drop cultures. Pharmacologic inhibition of cell migration by S1P1 blockage with FTY720 did not affect the presence of these cells, suggesting that migration of extra-thymic DPs does not require passage into blood. Transplantation of congenically marked thymi under the kidney capsule of thymectomized mice reconstituted T cells in the inguinal lymph node, but failed to reconstitute extra-thymic DPs. The discovery of two new, transcriptionally distinct subsets of extrathymic DPs reveals a novel, unexpected local escape mechanism from the thymus. Their association with blood vessels suggests possible angiotrophic functions.