T cells and inflammatory macrophages enter the CNS of MOG-immunized IL-23p19^−/− mice. (A) Phenotypic analysis of CNS-infiltrating cells in the brains of WT and IL-23p19^−/− mice at day 7 (before EAE onset) after immunization with MOG/CFA. (B) Intracellular cytokine staining of day 7 CNS-infiltrating cells from WT and IL-23p19^−/− mice after 18 h of stimulation with 100 μg/ml MOG peptide, all plots are gated on live CD4⁺ T cells. Data are representative of five separate experiments.

IL-23 promotes the expansion of IL-17 producing CD4⁺ (Th_IL-17) cells. (A) Intracellular IL-17 and IFN-γ production by CD4⁺ DLN cells isolated from PLP-immunized WT SJL mice, either immediately after ex vivo isolation, or cultured with PLP_139-151 peptide plus either rIL-12, rIL-23, or no added cytokine for 5 d. All plots are gated on live CD4⁺ T cells. (B) Mean percentage (±1 SD of error) of CD4⁺ T cells producing IFN-γ or IL-17 in response to 5 d of culture with IL-12 or IL-23. *, Student's unpaired t test, P < 0.02, averaged from eight separate experiments. (C) Intracellular IL-17 and IFN-γ production by CD4⁺ DLN cells isolated from MOG-immunized p40^−/− C57BL/6 either immediately after ex vivo isolation or cultured for 5 d with rIL-12 or rIL-23. All intracellular staining samples were restimulated with PMA/ionomycin for 4 h before analysis. All plots are gated on live CD4⁺ T cells and are representative of three independent experiments.

IL-23 drives a small population of naive T cells to produce IL-17 after in vitro activation. (A) Surface expression of CD62L on naive splenic DO11.10 × Rag^−/− T cells, either analyzed immediately after isolation (ex vivo) or on day 7 after in vitro anti-CD3/anti-CD28 stimulation plus IL-12 or IL-23. (B) Intracellular cytokine staining of CD4⁺ DO11.10 × Rag^−/− T cells on day 7 after in vitro anti-CD3/anti-CD28 stimulation plus IL-12 or IL-23. Cells were restimulated with PMA/ionomycin for 4 h before intracellular staining. (C) Naive DO11.10 × Rag^−/− T cells were parked in BALB/c recipients and OVA/CFA immunized. DLN cells were restimulated, PMA/ionomycin and intracellular stained for IL-17 and IFN-γ or (D) surface stained for CD44 and CD62L. All plots are gated on CD4⁺ KJ1-26⁺ cells. Results are representative of two experiments.

Gene expression analysis of IL-23 versus IL-12 driven T cells. (A) Quantitative analysis of mRNA expression levels of selected genes, comparing sorted CD4⁺ DLN cells isolated from PLP-immunized mice, either not cultured (ex vivo) or cultured with rIL-23 or rIL-12 for 10 d. (B) Quantitative analysis of mRNA expression levels of >200 selected genes, comparing total DLN cells isolated from PLP-immunized mice, cultured with either rIL-23 or rIL-12 for 10 d. All samples were activated with PMA/ionomycin for 4 h before RNA extraction and Taqman analysis. Results are normalized to a housekeeping gene, ubiquitin, and representative of two similar experiments.

IL-17–producing Th_IL-17 cells induce EAE. (A) PLP-primed DLN cells cultured with rIL-23 or rIL-12 for 10 d, labeled with CFSE (peak MFI = 4,654) and transferred (5 × 10⁶ cells/mouse) into recipient WT SJL mice. Cells were tracked at day 3 after immunization, with further gating on splenic CFSE⁺ CD4⁺ cells to determine IFN-γ and IL-17 production by intracellular staining. (B) PLP-primed DLN cells cultured with either rIL-23 (squares) or rIL-12 (triangles) for 10 d, and transferred into WT SJL mice as either purified CD4⁺ cells (3 × 10⁶ cells/mouse; n = 10/group; top), or as total DLN cells (7 × 10⁶ cells/mouse; n = 3/group; bottom), with mean EAE scores recorded. (C) Mean EAE scores of WT recipient mice (n = 5/group) passively transferred with graded numbers of Th_IL-17 cells: 1.2 × 10⁶ Th_IL-17 (closed squares), 3 × 10⁵ Th_IL-17 (closed triangles), 1.5 × 10⁵ Th_IL-17 (open circles), or 2.0 × 10⁴ Th_IL-17 (open diamonds). Results are summarized from two separate experiments. (D) Mean EAE scores of WT recipient mice (n = 5/group) passively transferred with either 5 × 10⁵ Th_IL-17 cells (open squares), 5 × 10⁵ Th1 cells (open diamonds), or cotransferred with Th_IL-17 plus Th1 cells (5 × 10⁵ of each; closed triangles), or Th_IL-17 plus sorted IFN-γ⁻ cells (5 × 10⁵ Th_IL-17 plus 2 × 10⁵ IFN-γ⁻ cells; closed circles). (C–D) Th_IL-17 cell numbers are calculated from intracellular cytokine staining analysis, with ∼3-5 × 10⁶ total DLN cells passively transferred per recipient mouse.

IL-17, not IFN-γ, contributes to EAE severity. (A) WT SJL mice actively immunized with PLP emulsified in CFA, and treated at day 7 with 200 μg anti–IL-17 (closed diamonds) or isotype control (open diamonds), n = 5/group. (B) WT SJL mice actively immunized with PLP emulsified in CFA (using suboptimal immunization strategy to give weaker disease), and treated at days 0 and 7 with 200 μg anti–IFN-γ (closed squares) or isotype control (open squares), n = 7/group. Plots are representative of three similar experiments.