Figure 8. CCR4 is required for medullary accumulation of post-positive selection DP and CD4SP thymocytes. (A) A maximum intensity projection of Ccr4+/+ (red) and Ccr4−/− (green) CD4SP thymocytes in live pCX-EGFP (blue) thymic slices. The boundary between cortex (C) and medulla (M) is indicated with a dashed line Video 1 shows corresponding two-photon imaging data. Bar, 100 µm. (B) Densities of Ccr4+/+ and Ccr4−/− CD4SP thymocytes in medulla and cortex (cells/µm3) were quantified from data as in A, and the ratio of medullary to cortical density was plotted for each dataset. Data represent three independent experiments with three time-lapse movies per experiment. **, P < 0.01 (paired Student’s t test). (C) Migration of WT thymocytes toward 100 nM CCL19 was assayed using in vitro Transwell chemotaxis assays. The migration index is the ratio of thymocytes migrating to the bottom chamber in response to CCL19 versus media alone. The horizontal line at 1 indicates no chemotaxis. Means + SEM compiled from three independent experiments, with three technical replicates per experiment. (D) Maximum intensity projections of Ccr4+/+ or Ccr4−/− DP CD69+ thymocytes migrating in pCX-EGFP (green) thymic slices. The boundary between cortex (C) and medulla (M) is outlined with a dashed line. Videos 2 and 3 show corresponding two-photon imaging data. Bar, 100 µm. (E) The ratios of medullary/cortical density for Ccr4+/+ and Ccr4−/− DP CD69+ cells were calculated from data, as in D. Means + SEM from three independent experiments with three time-lapse movies acquired for each group per experiment. (F) Representative confocal images of CD8 (green), CD4 (red), and CD69 (blue) on immunostained cryosections from Ccr4+/+ and Ccr4−/− thymi. White arrows indicate CD4+CD8+ DP thymocytes. Bar, 40 µm. (G) Nine medullary regions from three Ccr4+/+ and three Ccr4−/− thymi immunostained as in F were analyzed to calculate the mean number of DP CD69+ thymocytes per square millimeter. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (unpaired Student’s t test unless otherwise indicated). See also Fig. S1.
Figure 8.

CCR4 is required for medullary accumulation of post-positive selection DP and CD4SP thymocytes. (A) A maximum intensity projection of Ccr4+/+ (red) and Ccr4−/− (green) CD4SP thymocytes in live pCX-EGFP (blue) thymic slices. The boundary between cortex (C) and medulla (M) is indicated with a dashed line Video 1 shows corresponding two-photon imaging data. Bar, 100 µm. (B) Densities of Ccr4+/+ and Ccr4−/− CD4SP thymocytes in medulla and cortex (cells/µm3) were quantified from data as in A, and the ratio of medullary to cortical density was plotted for each dataset. Data represent three independent experiments with three time-lapse movies per experiment. **, P < 0.01 (paired Student’s t test). (C) Migration of WT thymocytes toward 100 nM CCL19 was assayed using in vitro Transwell chemotaxis assays. The migration index is the ratio of thymocytes migrating to the bottom chamber in response to CCL19 versus media alone. The horizontal line at 1 indicates no chemotaxis. Means + SEM compiled from three independent experiments, with three technical replicates per experiment. (D) Maximum intensity projections of Ccr4+/+ or Ccr4−/− DP CD69+ thymocytes migrating in pCX-EGFP (green) thymic slices. The boundary between cortex (C) and medulla (M) is outlined with a dashed line. Videos 2 and 3 show corresponding two-photon imaging data. Bar, 100 µm. (E) The ratios of medullary/cortical density for Ccr4+/+ and Ccr4−/− DP CD69+ cells were calculated from data, as in D. Means + SEM from three independent experiments with three time-lapse movies acquired for each group per experiment. (F) Representative confocal images of CD8 (green), CD4 (red), and CD69 (blue) on immunostained cryosections from Ccr4+/+ and Ccr4−/− thymi. White arrows indicate CD4+CD8+ DP thymocytes. Bar, 40 µm. (G) Nine medullary regions from three Ccr4+/+ and three Ccr4−/− thymi immunostained as in F were analyzed to calculate the mean number of DP CD69+ thymocytes per square millimeter. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (unpaired Student’s t test unless otherwise indicated). See also Fig. S1.

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