TGF-beta-treated antigen presenting cells suppress collagen- induced arthritis through the promotion of Th2 responses

Abstract

Collagen-induced arthritis (CIA) is mediated by self-reactive CD4(+) T cells that produce inflammatory cytokines. TGF-beta(2)-treated tolerogenic antigen-presenting cells (Tol-APCs) are known to induce tolerance in various autoimmune diseases. In this study, we investigated whether collagen-specific Tol-APCs could induce suppression of CIA. We observed that Tol-APCs could suppress the development and severity of CIA and delay the onset of CIA. Treatment of Tol-APCs reduced the number of IFN-gamma- and IL-17-producing CD4(+) T cells and increased IL-4- and IL-5-producing CD4(+) T cells upon collagen antigen stimulation in vitro. The suppression of CIA conferred by Tol-APCs correlated with their ability to selectively induce IL-10 production. We also observed that treatment of Tol-APCs inhibited not only cellular immune responses but also humoral immune responses in the process of CIA. Our results suggest that in vitro-generated Tol-APCs have potential therapeutic value for the treatment of rheumatoid arthritis as well as other autoimmune diseases.

Figure 1

Effect of Imm-APCs vs. Tol-APCs on Ag-specific CD4⁺ T cell responses. PECs derived from BALB/c WT mice were loaded with OVA protein with or without TGF-β₂. Tol-APCs or Imm-APCs were cultured with CFSE-labeled CD4⁺ T cells from OVA-primed DO11.10 mice. (A, B) After 72 h, the cultured cells were stained for proliferation of Ag-specific CD4⁺ T cells with anti-TCRβ and anti-CD4 and for intracellular cytokine staining with anti-IFN-γ. (C) IFN-γ production in the culture supernatants was measured by ELISA. The results represent the mean ± SEM (3-4 mice per group). Similar results were obtained in two independent experiments. ^*P < 0.05 versus Tol-APC treatment.

Figure 2

Treatment with Tol-APCs reduces the severity and inhibits the onset of CIA. To induce CIA, mice were immunized i.d. at the base of the tail with 100 µg of chicken CII emulsified with an equal volume of CFA. The mice were boosted by i.d. injection with 100 µg of CII in IFA. Seven days later, mice received i.v. injections of 1 × 10⁶ Imm-APC (○) or Tol-APC (■) or no APC transfer as the CIA control (●). (A) The clinical scores of arthritis in each group. Each paw was scored from 0 to 5 according to the severity of arthritis, with a maximal score of 20 per mouse. (B) The percentages of arthritic mice. Results are representative of three independent experiments with similar results. Bars show the mean ± SEM (6-8 mice per group). ^***P < 0.001, ^*P < 0.05 versus Tol-APCs-treated mice.

Figure 3

CII-specific T cell responses in Tol-APCs-treated and Imm-APCs-treated mice. Mice were immunized as previously described. Splenic cells were collected from CIA-induced mice on 45 days after the first immunization and re-stimulated in vitro with 100 µg/ml of CII. After 72 h, the cells were stained with anti-TCRβ, anti-CD4, anti-IFN-γ and anti-IL-17 mAbs or anti-IL-5 and anti-IL-4 mAbs for intracellular cytokine staining. (A) Dot plots show IFN-γ, IL-17, IL-5 and IL-4 secretion of gated CD4⁺ T cells. (B) Bars represent the percentage of the cytokine-secreting CD4⁺ T cells after re-stimulation for 72 h. (C) Cytokines in the culture supernatants were measured by ELISA. The results represent the mean ± SEM (6-8 mice per group). Similar results were obtained in three independent experiments. ^***P < 0.005, ^*P < 0.05 versus Tol-APCs-treated mice.

Figure 4

CII-specific antibody responses in Tol-APCs-treated and Imm-APCs-treated mice. Mice were immunized i.d. at the base of the tail with 100 µg of CII emulsified with an equal volume of CFA and boosted with 100 µg of CII in IFA on day 21. Seven days later, mice received i.v. injections of 1 × 10⁶ Tol-APCs or Imm-APCs. The levels of CII-specific total anti-IgG, -IgG1 and -IgG2a in the serum collected at day 45 were determined by ELISA. These results are representative of three independent experiments with similar results. ^***P < 0.005, ^*P < 0.05 versus Tol-APCs-treated mice.