Deletion of CCX-CKR does not affect regulatory T cells but leads to skewing of CD4⁺ T-cell responses from Th1 to Th17 and increased IL-23 levels. Mice were immunized for EAE and euthanized on day 12 postimmunization. (A) LN cells and splenocytes were harvested, cell-surface CD4 stained and then fixed/permeabilized overnight and stained for intracellular FoxP3. Cells were then analyzed by flow cytometry. Representative dot plots gating on lymphocytes by forward and side scatter are shown. Five mice per group were analyzed and a repeat experiment produced a similar dataset. Bar graphs showing the percentage (%) of CD4⁺ lymphocytes in draining lymph nodes and spleen-expressing FoxP3 are shown. (B) Leukocytes were harvested from spleen, draining LNs, and spinal cord and activated for 4 hours with PMA/ionomycin in the presence of GolgiStop. Cell-surface CD4 and intracellular IL-17 and IFNγ were then detected and cells analyzed by flow cytometry. Five mice per group were analyzed. Representative dot plots gated on CD4⁺ lymphocytes are shown. The proportion of CD4⁺ cells from spinal cord that have a Th17 phenotype is shown. Data are representative of the mean ± SEM (*P < .05). The ratio of Th1 to Th17 cells in draining LNs, spleen, and spinal cord is also shown. Data are representative of the mean ± SEM (*P < .05). (C) Spleen and draining lymph node protein lysates were prepared from female wild-type and CCX-CKR^−/− mice on day 12 postimmunization for MOG_35-55 EAE and IL-23, IL-6 and TGFβ protein levels assessed by ELISA. Data points show the mean amount of each of these proteins per milligram of tissue homogenized ± SEM (n = 9 mice per group per time point; **P < .01).