CCR4 is expressed on post-positive selection thymocytes, whereas CCR4 ligands are expressed by thymic DCs. (A) Ccr4 mRNA expression levels in FACS-purified thymocyte and lymphocyte subsets, normalized to expression in DP CD69⁺ cells, as determined by qRT-PCR. Means + SEM for one of three representative experiments with three technical replicates each. (B) Flow cytometric profiles of cell surface CCR4 levels on thymocytes and lymph node T cell subsets. Representative of three mice per group. (C and D) mRNA expression levels of the CCR4 ligands Ccl22 and Ccl17 in FACS-purified thymic stromal subsets, normalized to expression in Sirpα⁺ DCs, as determined by qRT-PCR. Means + SEM for one of two representative experiments with two technical replicates each. (E and F) Fold change in the number of cells of the indicated subsets migrating toward 100 nM CCL22 (E) or 100 nM CCL17 (F) relative to media alone in Transwell chemotaxis assays. Means + SEM for one of three representative experiments with three technical replicates each. (G) Immunostaining of CD11c (red), Sirpα (green), and DAPI (blue) in thymic cryosections from C57Bl6/J mice. The boundary between cortex (C) and medulla (M) is indicated with a dashed line. White arrows indicate Sirpα⁺ DCs; black arrows indicate globular Sirpα⁺ CD11c⁻ cells, presumably macrophages. Bar, 50 µm. Representative of two mice analyzed in two separate experiments. *, P < 0.05; **, P < 0.01 (unpaired Student’s t test).