Fig. 3.

In vivo labeling identifies emigrating CD4 SP thymocytes. (A) Flow cytometric detection of intravascularly CD4PE-labeled DP cells in the thymus of S1P1A transgenic (A) versus control (−) mice. Thymocytes were isolated 5 minutes after CD4PE antibody injection. Bar graph shows the average percent of CD4PE-positive cells in transgenic mice that were also CD8-positive, from n= 3 mice. Circles represent individual mice. (B) Immunofluorescence of thymic section from an S1P1A transgenic mouse pretreated with CD4PE. Panels 1–12 show a confocal z-series through an emigrating DP cell (0.3μm z step). Single color panel shows CD8 staining, indicating cortical location of transmigration. Scale bars represent 10 (top) or 5 (bottom) μm. Data are representative of at least 3 mice analyzed in 3 experiments. (C) Flow cytometric detection of intravascularly CD4PE-labeled SP cells in the thymus of non-transgenic mice and their absence after 12 h FTY720 treatment. Bars in right graph show mean of 3 experiments (n=3 mice). (D) RAG-GFP intensity and CD31 staining on the indicated CD4PE-labeled cells. Left histogram plot shows GFP fluorescence in intravascularly CD4PE-labeled cells in thymus, mature CD62L^hi Qa2^int CD4 SP thymocytes, and CD4 T cells in blood, and numbers indicate % GFP^hi for each cell type. Middle graph shows summarized data for 3 mice aged 5 to 6 weeks. Right histogram plot shows CD31 staining with inset fluorescent intensities for the indicated thymic populations and RAG-GFP^hi blood recent thymic emigrants (RTE). Data are representative of 5 mice analyzed in 5 experiments. (E) RAG-GFP intensity of CD69^−/− (blue) and CD69^+/− (red) mature CD4 SP and IV CD4PE⁺ thymocytes. Bottom bar graph shows ratios of GFP intensity comparing mature CD4 SP and IV CD4PE⁺ thymocytes for individual mice. These data represent 7 mice analyzed in 7 experiments.