Figure 3. Ccl17 deficiency affects T cell distributions.

(A) Quantification of PE-CD4⁺ T cells and APC-Tregs accumulating in atherosclerotic aortas of high-fat diet–fed Ccl17^+/+Apoe^–/– and Ccl17^E/EApoe^–/– mice 3 days after adoptive transfer, using enzymatic digestion and FACS analysis. Representative dot plots depict frequencies of labeled cells among CD45⁺ aortic cells. Data points represent frequencies of transferred aortic cells in individual mice; horizontal bars denote mean of all mice. (B) Transwell migration of CD4⁺ T cells toward Ccl17^E/+ or Ccl17^E/E BMDCs was quantified by FACS analysis (n = 5). (C) Absolute numbers of CD4⁺ T cells recruited to air pouches injected with PBS (ctrl, n = 8) or recombinant mouse CCL17 (50 ng/ml, n = 9) were quantified in lavage fluid after 4 hours. (D and E) Flow cytometric analysis of CD3⁺CD4⁺ T cells (D) and CD4⁺Foxp3⁺CD25⁺ Tregs (E) in LNs of Ccl17^+/+ and Ccl17^E/E mice employing indicated surface markers. Representative dot plots as well as relative and absolute numbers of Tregs are shown; numbers in dot plots are percentage of CD4⁺ events. Data points represent frequencies of cells in individual mice; horizontal bars, mean of all mice. (F) Foxp3 mRNA expression in LNs of Ccl17^+/+Apoe^–/– and Ccl17^E/EApoe^–/– mice fed a high-fat diet (n = 3 each). (G) Detection of Foxp3⁺GFP⁺ cells (arrows, both panels) in atherosclerotic plaques of Ldlr^–/– mice reconstituted with Foxp3gfp.KI BM after 9 weeks of high-fat diet; cell nuclei were counterstained by DAPI (blue, lower panel); dotted lines demarcate lesional area. Scale bars: 50 μm. (H) Foxp3 mRNA expression in atherosclerotic aortas of Ccl17^+/+Apoe^–/– and Ccl17^E/EApoe^–/– mice on high-fat diet (n = 3 each). (I) Quantification of CD4⁺CD25⁺Foxp3⁺ Tregs in atherosclerotic aortic segments of Ccl17^+/+Apoe^–/– and Ccl17^E/EApoe^–/– mice on high-fat diet using enzymatic digestion and FACS analysis. *P < 0.05.