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. 2005 Jan 17;201(2):233-40.
doi: 10.1084/jem.20041257.

IL-23 drives a pathogenic T cell population that induces autoimmune inflammation

Claire L Langrish  1 , Yi Chen, Wendy M Blumenschein, Jeanine Mattson, Beth Basham, Jonathan D Sedgwick, Terrill McClanahan, Robert A Kastelein, Daniel J Cua
Affiliations

IL-23 drives a pathogenic T cell population that induces autoimmune inflammation

Claire L Langrish et al. J Exp Med. .

Abstract

Interleukin (IL)-23 is a heterodimeric cytokine composed of a unique p19 subunit, and a common p40 subunit shared with IL-12. IL-12 is important for the development of T helper (Th)1 cells that are essential for host defense and tumor suppression. In contrast, IL-23 does not promote the development of interferon-gamma-producing Th1 cells, but is one of the essential factors required for the expansion of a pathogenic CD4(+) T cell population, which is characterized by the production of IL-17, IL-17F, IL-6, and tumor necrosis factor. Gene expression analysis of IL-23-driven autoreactive T cells identified a unique expression pattern of proinflammatory cytokines and other novel factors, distinguishing them from IL-12-driven T cells. Using passive transfer studies, we confirm that these IL-23-dependent CD4(+) T cells are highly pathogenic and essential for the establishment of organ-specific inflammation associated with central nervous system autoimmunity.

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Figures

Figure 1.
Figure 1.
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.
Figure 2.
Figure 2.
IL-23 promotes the expansion of IL-17 producing CD4 + (ThIL-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 PLP139-151 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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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.
Figure 5.
Figure 5.
IL-17–producing ThIL-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 ThIL-17 cells: 1.2 × 106 ThIL-17 (closed squares), 3 × 105 ThIL-17 (closed triangles), 1.5 × 105 ThIL-17 (open circles), or 2.0 × 104 ThIL-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 ThIL-17 cells (open squares), 5 × 105 Th1 cells (open diamonds), or cotransferred with ThIL-17 plus Th1 cells (5 × 105 of each; closed triangles), or ThIL-17 plus sorted IFN-γ− cells (5 × 105 ThIL-17 plus 2 × 105 IFN-γ− cells; closed circles). (C–D) ThIL-17 cell numbers are calculated from intracellular cytokine staining analysis, with ∼3-5 × 106 total DLN cells passively transferred per recipient mouse.
Figure 6.
Figure 6.
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.
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