Accumulation of CCR6⁺ T cells in the CNS infiltrate during the course of EAE. (A) FACS analysis of cell surface markers CD25 and CCR6 on CD4⁺ T-cell populations of PBMCs and CNS infiltrate. Lymphocytes of the peripheral blood (top row) and of the CNS infiltrate (bottom row) were stained with antibodies directed against CD4, CD25, and CCR6. Samples were taken from naive mice (left panels), from mice that developed active EAE with a clinical score of 4 (middle panels), or from mice that surpassed the state of active EAE and already reached remission (score 0; right panels). Dot plots are shown for the populations gated on CD4⁺ cells and display surface staining of CD25 versus CCR6. Numbers represent the percentages of CD4⁺ cells in each quadrant. One of 4 independent experiments is shown. (B) FoxP3 expression. mRNA expression is shown for FACS populations isolated from the CNS infiltrate of mice with active EAE and from mice in remission. Expression was determined by real-time RT-PCR and is shown for the CCR6^- and CCR6⁺ subsets of CD25^-CD4⁺ T cells (□) and of CD25⁺CD4⁺ T cells (▦). Relative expression is shown in reference to the expression level of the CD25^-CCR6^- subset. Expression of CD25^-CD4⁺ and CD25⁺CD4⁺ splenocytes derived from naive mice are shown as control. Each experiment contained RNA from cells obtained by FACS that were pooled from 9 to 10 mice per group. (C) GITR-mediated abrogation of suppressor activity in CNS infiltrates. CNS-infiltrating lymphocytes isolated from mice with active EAE (score 4) and from mice in remission (score 0) were stimulated with anti-CD3 in the absence (□) or presence of anti-GITR (▦). Proliferation was determined after addition of ³H-thymidine and is expressed as fold increase of the proliferation detected in the absence of anti-GITR. One of 2 independent experiments is shown each with cells from 3 to 5 mice per group. (D) Percentage of CCR6⁺CD25^- and CCR6⁺CD25⁺ cells in blood and CNS infiltrate. Bars indicate percentage of CCR6⁺ cells of the CD25^-CD4⁺ and the CD25⁺CD4⁺ subset within the PBMCs (□) or the CNS infiltrate (▦). P values are represented by asterisks (single asterisk, P < .05; double asterisk, P < .01) and were calculated according to the Student t test.