CCR4 and CCR7 differentially regulate thymocyte subset localization with distinct outcomes for central tolerance

Abstract Central tolerance is established as T cells mature in the thymus by the combined effects of clonally deleting autoreactive thymocytes or inducing their diversion to the Treg lineage. To ensure T cell tolerance to diverse self-antigens, thymocytes must efficiently enter the medulla and scan heterogeneous antigen presenting cells, which display the majority of the peptidome in a sparse mosaic. While it is widely accepted that CCR7 contributes to central tolerance by promoting thymocyte medullary localization and rapid migration therein, the contribution of CCR4 to these processes remains unclear. For example, it is not known whether CCR4 is simply redundant to CCR7 in promoting medullary entry or if it plays a distinct role in central tolerance. Here, we report that early post-positive selection thymocytes undergo chemotaxis towards CCR4 ligands, while progressively mature cells migrate towards CCR7 ligands. Synchronized positive selection assays reveal that CCR4 is upregulated within hours of positive selection signaling, while CCR7 upregulation is delayed. 2-photon microscopy shows that CCR4 facilitates entry of early post-positive selection cells into the thymic medulla, while CCR7 promotes medullary accumulation of mature subsets. These findings suggest that CCR4 and CCR7 have non-redundant roles in negative selection. Consistent with this possibility, flow cytometric analysis reveals that CCR4 deficiency results in a selective decline in early-stages of negative selection, while CCR7 deficiency impairs only late-stage negative selection. Our data support a new model in which CCR4 and CCR7 mediate medullary entry of different thymocyte subsets and play distinct roles in enforcing central tolerance.