Role of CD25+ Dendritic Cells in the Generation of Th17 Autoreactive T Cells in Autoimmune Experimental Uveitis

Dongchun Liang, Aijun Zuo, Hui Shao, Willi K. Born, Rebecca L. O’Brien, Henry J. Kaplan and Deming Sun
J Immunol June 1, 2012, 188 (11) 5785-5791; DOI: https://doi.org/10.4049/jimmunol.1200109

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Injection of B6 mice with anti-mouse CD25 Ab (PC61) decreases the Th17 autoreactive T cell response. (A) Splenic T cells from IRBP1–20/CFA-immunized mice, with or without prior PC61 treatment, were enriched by passage through nylon wool and stimulated for 48 h with an optimal dose of IRBP1–20 (10 μg/ml) under Th1- or Th17-polarized conditions. The activated T cells were separated by Ficoll-gradient centrifugation on day 3, cultured under the same polarized conditions for an additional 5 d, and intracellularly stained with PE-conjugated anti–IFN-γ Abs and FITC-conjugated anti–IL-17 Abs, followed by FACS analysis. (B) IFN-γ and IL-17 levels in the culture supernatants were measured by ELISA after the 48-h incubation with peptide. (C) IRBP-specific T cells (2 × 106/mouse) from untreated and PC61-treated IRBP-immunized mice activated under Th1- or Th17-polarized conditions were adoptively transferred to syngeneic B6 mice. Pathologic examination was conducted 10 d after disease induction. A set of representative pathologic slides is shown (left panels) along with summarized in vivo results (right panel). H&E staining. Original magnification ×200. (D) Responder T cell numbers were evaluated by LDA, as detailed in Materials and Methods. The results shown are representative of those from more than five experiments.

  • FIGURE 2.
    FIGURE 2.

    Phenotypic change of T cells in PC61-injected mice. B6 mice were randomly separated into two groups (n = 8) and immunized with IRBP1–20/CFA, with or without prior treatment with PC61. After 13 d, splenic T cells were enriched and stimulated with the immunizing peptide. The phenotype of the T cells was analyzed either before (A–C) or after (D–F) in vitro stimulation. Effect of PC61 injection on CD25+CD3+ cells (A), Foxp3+ cells (B), and γδ T cells (E). (D–F) After in vitro stimulation with the immunizing Ag and expansion under Th17-polarized conditions, the activated T cells were stained with the indicated Abs and analyzed by flow cytometry. The experiments were repeated more than five times, and the results of one representative experiment are shown.

  • FIGURE 3.
    FIGURE 3.

    Criss-cross tests showing that dysfunction of splenic APCs accounts for the decreased generation of IL-17+ IRBP-specific T cells in PC61-treated mice. (A) Responder T cells from immunized PC61-treated or nontreated mice were stimulated in vitro for 48 h with IRBP1–20 peptide in the presence of splenic APCs from PC61-treated or nontreated mice. The percentage of IL-17+ T cells was measured. (B) IL-17 levels in the culture supernatants after a 48-h incubation with peptide and APCs were assessed by ELISA.

  • FIGURE 4.
    FIGURE 4.

    CD25+CD11c+ cells are more effective than are CD25−CD11c+ cells in stimulating the activation of IL-17+ IRBP-specific T cells and γδ TCR+ T cells. (A and B) Splenic cells of immunized mice, with or without PC61 injection, were stained for expression of CD11c and/or CD25. (C) CD25+CD11c+ and CD25−CD11c+ DCs were separated using magnetic beads. (D and E) Splenic T cells isolated from immunized B6 mice were stimulated with the immunizing peptide IRBP1–20 in the presence of CD25−CD11c+ (left panels) or CD25+CD11c+ (right panel) DCs for 5 d. The activated T cells were stained for the expression of IL-17 (D) and IFN-γ (E). (F) ELISA assay. Culture supernatant of immunized splenic T cells was tested for IL-17 production after a 48-h stimulation with the immunizing peptide IRBP1–20 in the presence of either CD25−CD11c+ or CD25+CD11c+ DCs.

  • FIGURE 5.
    FIGURE 5.

    Restoration of the Th17 response by addition of activated γδ T cells to functionally defective T cells. In vivo-primed T cells from IRBP1–20/CFA-immunized mice with (B, C) or without (A) prior treatment with PC61 were stimulated for 5 d with immunizing peptide under Th1- or Th17-polarized conditions (A, B) or after addition of 2% of activated γδ T cells [2 × 104/well (C)]. Activated T cells were intracellularly stained for IFN-γ and IL-17 and analyzed by FACS for numbers of IL-17+ or IFN-γ+ T cells. (D) IFN-γ and IL-17 levels in the culture supernatants at 48 h were assessed by ELISA.

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