Neutralization of CCL21 in vivo reduces Th17 responses and delays EAE onset. (A) Mice were immunized for EAE and injected intraperitoneally with 500 μg of protein A–purified rabbit anti-CCL21 antisera or 500 μg of protein A–purified normal rabbit IgG (NRIgG) on days 1 and 6 postimmunization. On day 9 postimmunization, spleens were harvested and intracellular IL-17A and IFNγ detected in CD4⁺ lymphocytes after 4 hours activation in the presence of PMA/ionomycin in the presence of GolgiStop. Representative dot plots showing intracellular cytokines gated on CD4⁺ lymphocytes are presented along with data for Th1:Th17 from each mouse. The proportion of CD4⁺ cells that express both IL-17A⁺ and IFNγ⁺ was also quantified. Data shown are the mean ± SEM (**P < .01; *P < .05). (B) Mice were administered with either 500 μg of protein A–purified polyclonal anti-CCL21 antibodies or protein A–purified normal rabbit IgG via intraperitoneal injection the day before immunization, and on days 5 and 10 postimmunization. Mice were then monitored until disease onset. Data are pooled from 2 independent experiments with 5-8 mice in each group in each experiment. The mean day of disease onset of each mouse in each group is shown ± SEM (***P < .001; **P < .01).