Figure 1: Th17 cell recruitment to the CNS is temporally regulated by CCR6 and CCR2.

(a) Quantitative PCR of Ccr6 and Ccr2 transcript in CCR6⁺ and CCR6⁻ subsets of CD4⁺IL-17A^+/ex (currently, or previously Th17) cells (CD3⁺CD4⁺CD44^hiIL-17AeYFP⁺–B6.Il17a^CreRosa26^eYFP mice) and Tregs (CD3⁺CD4⁺Foxp3GFP⁺–B6.Foxp3^GFP mice) from the spleen/draining lymph node (dLN) of 5–6 mice d10 post MOG/CFA immunization. Data presented relative to Rplp0 (mean±s.d.). (b) Representative flow cytometric analysis of CCR6/CCR2 staining on naive CD4⁺ (CD3⁺CD4⁺CD44^lo), Th1 (CD3⁺CD4⁺CD44^hiIL-17A⁻IFNγ⁺), Th17 (CD3⁺CD4⁺CD44^hiIL-17A⁺) and Tregs (CD3⁺CD4⁺Foxp3⁺) from the spleen of B6 mice d10 post MOG/CFA immunization. Data are representative of three independent experiments with n=3–4 mice per experiment. (c) Transwell chemotaxis to CCL20 and CCL2 by indicated T-cell subsets from d10 MOG/CFA-immunized B6 mice. Th17 cells from B6.Ccr6^−/− and B6.Ccr2^−/− mice served as CCL20 and CCL2 controls, respectively. Data are representative of two independent experiments with n=4 mice per experiment. (d) Representative flow cytometric analysis of CCR6/CCR2 staining on Th17 cells (CD3⁺CD4⁺CD44^hiIL-17A⁺) in the dLN, spleen and CNS on d5, 10 and 15 post EAE induction. Data are representative of four independent experiments with n=4–6 mice per timepoint. (e,f) EAE-immunized B6 mice were administered 100 μg of CCL2_ala (scrambled peptide control; n=5), CCL20_6–70 (CCR6 antagonist; n=4) or CCL2_9–76 (CCR2 antagonist; n=5) i.p. on days 2, 4, 6 and 7 (e) or days 8, 10, 12 and 13 (f). CNS-infiltrating Th17 cells were quantified 24 h after the final antagonist treatment. (g) Schematic of Th17 cell transfer system. (h) Number of transferred Th17 cells (CD3⁺CD4⁺IL-17A⁺CD45.2⁺) in CNS 48 h post transfer from CCL20_6–70 (n=5), CCL2_9–76 (n=5) or CCL2_ala (n=5) treated B6.Ly5.1 recipients pre-immunized for EAE 5 (left) or 15 (right) days prior. (i) Number of transferred CD45.2⁺ B6 (n=5) or B6.Ccr6^−/− Th17 cells in CNS 48 h post transfer of CCL2_ala (n=6)- or CCL2_9–76 (n=6)-treated B6.Ly5.1 recipients pre-immunized for EAE 15 days prior. (j) Number of transferred CD45.2⁺ Th17 cells in CNS 48 h post transfer of PBS (n=5)- or anti-CCL2 (n=5)-treated B6.Ly5.1 recipients pre-immunized for EAE 15 days prior. (c,e,f,h,i,j) Data are presented as mean±s.e.m. (e,f,h–j) *P≤0.05, **P≤0.01; (e,f,h) one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test relative to control CCL2_ala-treated group; (i) one-way ANOVA with Bonferroni multiple comparisons test; (j) unpaired two-tailed Student’s t-test.