Functional characterization of CCR6⁺ Treg cells. (A) Chemotactic response of CD25^-CD4⁺ and CD25⁺CD4⁺ T cells to CCL20. The chemotactic response of sorted populations of CD25^-CD4⁺ (left panel) and CD25⁺CD4⁺ lymph node cells (right panel) was tested in a migration assay. The cells were incubated with either medium alone, with 1 μg/mL of the CCR5 ligand CCL4, or with1 μg/mL of the CCR6 ligand CCL20. After 3 hours the number of migrated cells was determined by FACS analysis. Migration is expressed as chemotactic index and was calculated by the ratio of cells, migrated in the presence of chemokine, to the number of cells that spontaneously migrated with medium alone. One of 3 independent experiments is shown. (B) Depletion of CCR6⁺ cells abrogates CCL20-induced migration of CD25⁺CD4⁺ T cells. The experiment was carried out as described for panel A, except that populations of CD25⁺CD4⁺ splenocytes were used, in which the CCR6⁺ subset was either still present or had been removed prior to the experiment by MACS. (C) CCR6⁺CD25⁺ T cells up-regulate IL-10 expression on restimulation in vitro. Using FACS-sorted cells, RNA from CD25⁺CCR6⁺, CD25⁺CCR6^-, and as control of CD25^-CD4⁺ T cells was analyzed for IL-10 expression by real-time RT-PCR. RNA was prepared either from freshly isolated cells (left panel) or after stimulation for 4 hours with plate-bound αCD3 (right panel). The relative expression is shown in reference to the IL-10 mRNA level of CD25^- control cells. One of 4 independent experiments is shown each with pooled RNA from 8 to 10 animals.