CCR6⁺CD25⁺CD4⁺ cells exhibit phenotypic and functional characteristics of Treg cells. (A) FACS analysis of markers characteristic for Treg cells. Four-color staining was carried out with lymph node cells from naive BALB/c mice. Cells were stained with antibodies specific for CD4, CD25, and CCR6 together with antibodies specific for either CD45RB, CD152, GITR, or CD122. Histograms are shown for the 4 CD4⁺ T-cell subsets CD25⁺CCR6⁺ (i), CD25⁺CCR6^- (ii), CD25^-CCR6⁺ (iii), and CD25^-CCR6^- (iv). Plots are generated by gating on the respective markers. Staining of CD152 was carried out intracellularly; gray areas represent here the isotype control. Vertical lines are inserted as reference for relative shifts. (B) Expression of CD103 and CCR6 on the CD25⁺CD4⁺ T-cell subsets. The dot plot shows the staining CD103 versus CCR6 for the population of lymph node cells gated on CD25⁺CD4⁺ cells. Numbers in each quadrant indicate the percentage fraction of the CD25⁺CD4⁺ T-cell population. Plots in panels A and B are representative for 3 independent experiments with cells pooled from 2 to 5 animals. (C) Expression of FoxP3. CD25^-CCR6^-, CD25^-CCR6⁺, CD25⁺CCR6^-, and CD25⁺CCR6⁺ lymph node cells were isolated by FACS and expression levels of FoxP3 were determined by real-time RT-PCR. CCR6^- cells are represented by □, CCR6⁺ cells by ▦. Expression was determined in reference to HPRT and expression levels of FoxP3 are shown as fold increase compared to CD25^-CCR6^- T cells. The plot represents one of 4 independent experiments each with cells pooled from 8 to 10 mice. (D) Proliferative in vitro response of CCR6⁺CD25⁺ T cells. CCR6⁺CD25⁺ (▦) and CCR6^-CD25⁺ CD4⁺ T cells (□) were stimulated in vitro with anti-CD3 and radiated APCs in the absence or presence of 20 U/mL IL-2. Proliferation was determined with ³H-thymidine. One of 2 independent experiments with cells pooled from each of 10 mice is shown. (E) Suppressive capacity. The ability to suppress proliferation of CD25^-CD4⁺ splenocytes was tested with FACS populations of CD25⁺CD4⁺ lymph node cells differing in their CCR6 expression (left panel) and with CCR6⁺CD4⁺ lymph node cells differing in CD25 expression (right panel). CD25^-CD4⁺ splenocytes were incubated in the presence of anti-CD3 and radiated APCs with titrated amounts of CCR6^-CD25⁺CD4⁺ T cells (○ and dashed line) or CCR6⁺CD25⁺CD4⁺ T cells (• and solid line). Proliferation was determined with ³H-thymidine. The x-axis of the line plot indicates the ratio of CD25⁺CD4⁺ regulatory cells to CD25^-CD4⁺ cells. Bars of the bar chart represent proliferation of CD25^-CD4⁺ splenocytes alone and of CD25^-CD4⁺ splenocytes incubated with CCR6⁺CD25⁺ or with CCR6⁺CD25^- T cells at a ratio of 0.33. One of 3 independent experiments each with cells pooled from 8 to 10 mice is shown.