Ccr4 And Ccr7 Make Distinct Contributions To The Spatial Distribution And Negative Selection Of Different Thymocyte Subsets

Abstract Central tolerance mechanisms, including clonal deletion and Treg selection, are required to remove autoreactive T cells generated during thymocyte differentiation. To ensure T cell tolerance to diverse self-antigens, the majority of the peptidome is displayed in a sparse mosaic by medullary thymic epithelial cells and dendritic cells in the medulla. Thus, thymocytes must efficiently enter the medulla and scan multiple antigen presenting cells to ensure complete tolerance. We and others have previously reported that the chemokine receptors CCR4 and CCR7 are important regulators of medullary enrichment, rapid motility, and negative selection of post-positive selection thymocytes; however, it is not known whether CCR4 and CCR7 play distinct or redundant roles 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 experiments reveal that CCR4 is upregulated within hours of receiving a positive selection signal, while CCR7 upregulation is delayed for about 24h. 2-photon microscopy reveals that CCR4 facilitates entry of early post-positive selection cells into the thymic medulla. These findings suggest that CCR4 and CCR7 promote distinct stages of negative selection. Consistent with this possibility, flow cytometric analysis reveals that CCR4 deficiency results in a selective decline in early-stage negative selection, while CCR7 deficiency impairs only late-stage negative selection. Altogether, our data suggest a new model in which CCR4 and CCR7 mediate medullary entry of distinct thymocyte subsets and contribute differentially to central tolerance.