CCR6 expression of CD25⁺CD4⁺ T cells. (A) Gene array analysis of CD25⁺CD4⁺ and CD25^-CD4⁺ lymph node cells. mRNA expression levels are shown for preparations derived from CD4⁺CD25^- and CD4⁺CD25⁺ lymphocyte populations (via MACS) isolated from SJL/J mice. Diagonal lines indicate 3-fold overexpression; dots representing CTLA-4, GITR, and CCR6 are indicated by arrows. (B) Comparison of CCR5 and CCR6 expression on CD4⁺ lymph node cells. Cells from lymph nodes of naive BALB/c mice were triple-stained with antibodies directed against CD4, CD25, and either CCR6 (left panel) or CCR5 (right panel) and analyzed by FACS. The dot plots represent cells gated on CD4 expression. The numbers in each quadrant indicate the percentage of cells within the CD4⁺ population. One of 3 independent experiments with cells pooled from 2 to 5 animals is shown. (C) Relative fraction of CCR6⁺CD4⁺ T cells in lymph nodes, spleens, and PBMCs. Percentage of cells was determined by FACS analysis with cells from naive mice as described for Figure 1B. □ represents the percentage of CCR6⁺ cells within the population of CD4⁺CD25^- T cells; ▦, their fraction within the population of CD4⁺CD25⁺ T cells. Plot shows mean values from 7 to 9 independent experiments, P values are represented by asterisks (^**P < .01) and were calculated according to the Student t test.