Distinct Role of IL-27 in Immature and LPS-Induced Mature Dendritic Cell-Mediated Development of CD4+ CD127+3G11+ Regulatory T Cell Subset

Abstract

Interleukin-27 (IL-27) plays an important role in regulation of anti-inflammatory responses and autoimmunity; however, the molecular mechanisms of IL-27 in modulation of immune tolerance and autoimmunity have not been fully elucidated. Dendritic cells (DCs) play a central role in regulating immune responses mediated by innate and adaptive immune systems, but regulatory mechanisms of DCs in CD4⁺ T cell-mediated immune responses have not yet been elucidated. Here we show that IL-27 treated mature DCs induced by LPS inhibit immune tolerance mediated by LPS-stimulated DCs. IL-27 treatment facilitates development of the CD4⁺ CD127⁺3G11⁺ regulatory T cell subset in vitro and in vivo. By contrast, IL-27 treated immature DCs fail to modulate development of the CD4⁺CD127⁺3G11⁺ regulatory T cell sub-population in vitro and in vivo. Our results suggest that IL-27 may break immune tolerance induced by LPS-stimulated mature DCs through modulating development of a specific CD4⁺ regulatory T cell subset mediated by 3G11 and CD127. Our data reveal a new cellular regulatory mechanism of IL-27 that targets DC-mediated immune responses in autoimmune diseases such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE).

Keywords: IL-27; dendritic cell; immune tolerance; immunotherapy; regulatory T cell.

Figure 1

Expression of T_reg-associated molecules on MOG-primed CD4⁺ T cells co-culture with immature DCs (Thin line) or IL-27-treated immature DCs (Thick line) pulsed with MOG peptide in vitro. C57 BL/6J mice were immunized with MOG (200 μg)/Quil A (20 μg) / KLH (20 μg)/per mouse at day 0. Splenocytes were harvested at day 10. CD4⁺ T cells were then isolated using mouse CD4⁺ T cell subset column kit (R and D Systems). CD4⁺ T Lymphocytes were re-stimulated with MOG peptide (0.1 μM) and IL-2 (1 ng/ml) for 72 h. Cells were then stained by anti-mouse CD25 (A), CD127 (B), FoxP3 (C), GITR (D), and 3G11 (E) antibodies. Protein expression of T_reg-associated molecules on CD4⁺ T cells is shown. Error bars indicated in this figure represent mean and SD of triplicate determinations of mean fluorescence intensity (MFI) of T_reg-associated molecule expression on CD4⁺ T cells (n = 3, t test, P_A = 0.4198; P_B = 0.4450; P_C = 0.6047; P_D = 0.8372; P_E = 0.2523; NS, no significant difference).

Figure 2

Protein expression of T_reg-associated molecules on MOG-primed CD4⁺ T cells incubated with LPS-induced mature DCs or IL-27-treated mature DCs pulsed with MOG peptide in vitro. Bone marrow-derived dendritic cells were stimulated with LPS (1 μg/ml) for 24 hrs. LPS-stimulated DCs were also simultaneously incubated with IL-27 (20 ng/ml) (Thick line) for 72 hrs or had no IL-27 treatment (Dot line). DCs were then co-cultured with MOG-primed CD4⁺ T cells as shown in Figure 1. Protein expression of CD25 (A), CD127 (B), FoxP3 (C), GITR (D), and 3G11 (E) on CD4⁺ T cells is shown. Error bars indicated in this figure represent mean and SD of MFI of T_reg-associated molecules expressing on CD4⁺ T cells in three independent experiments (n = 3, t test, P_A = 0.8809; P_B = 0.3012; P_C = 0.2879; P_D = 0.7744; P_E = 0.7549; NS, no significant difference).

Figure 3

IL-27 treatment facilitates LPS-induced mature DCs-mediated development of CD4⁺CD127⁺3G11⁺ T_reg subset in vitro. Bone marrow-derived DCs shown in Figures 1, 2 were treated with IL-27 (20 ng/ml, 72 h) or/and LPS (1 μg/ml, 24 h) or without LPS or IL-27 incubation. DCs were then co-cultured with MOG-primed CD4⁺ T cells indicated in Figures 1, 2 for 72 h. CD4⁺CD25⁺FoxP3⁺GITR⁺ cells were gated. The frequency of CD127⁺3G11⁺ T_regs is shown. Isotype control is CD4⁺ T cells incubated with isotype control antibodies. Error bars shown in this figure represent mean and SD of frequency of CD4⁺CD127⁺3G11⁺ T_regs in three independent experiments (n = 3, t test, P _{(DC, DC+IL−27)} = 0.1357; P_{(DC+LPS, DC+LPS+IL−27)} = 0.0002).

Figure 4

i.v. transfer of IL-27-treated mature DCs induced by LPS inhibits immune tolerance mediated by LPS-stimulated DCs in vivo. C57 BL/6J mice were immunized with MOG peptide (200 μg/per mouse) and CFA. Immature dendritic cells (DCs) were incubated with IL-27 (DCs+IL-27) or LPS (DCs+LPS) or both IL-27 and LPS (DCs+LPS+IL-27). Mice in control group were i.v. transferred with PBS. EAE was then induced and shown by clinical score. Error bars in this figure represent mean and SEM of triplicate determinations of EAE clinical score in one experiment (n = 3, two-way ANOVA test, P _{(DC, DC+IL−27)} = 0.7960; P_{(DC+LPS, DC+LPS+IL−27)} = 0.0001; NS, no significant difference).

Figure 5

IL-27-treated immature DCs do not affect expression of T_reg-associated molecules on CD4⁺ T cells ex vivo. Spleen cells were isolated from mice treated with PBS (Shade) or DCs (Dot line) or DCs+IL-27 (Thin line) indicated in Figure 4. T lymphocytes were re-stimulated by MOG peptide (0.1 μM) and IL-2 (1ng/ml) for 72 h. The expression of CD25 (A), CD127 (B), FoxP3 (C), GITR (D), and 3G11 (E) on CD4⁺ T cells is shown. Error bars demonstrated in this figure represent mean and SD of MFI of T_reg-associated molecules present on CD4⁺ T cells in three independent experiments (n = 3, t test, P_A = 0.1944; P_B = 0.1476; P_C = 0.2879; P_D = 0.7744; P_E = 0.7549; NS, no significant difference).

Figure 6

IL-27-treated mature DCs induced by LPS do not affect expression of T_reg-associated molecules on CD4⁺ T cells ex vivo. LPS-stimulated DCs (Thin line) or IL-27-treated mature DCs induced by LPS (Thick line) were i.v. transferred into mice shown in Figure 4. Spleen cells were isolated and re-stimulated with MOG peptide (0.1 μM) and IL-2 (1 ng/ml) for 72 hrs. The expression of CD25 (A), CD127 (B), FoxP3 (C), GITR (D), and 3G11 (E) on CD4⁺ T cells is shown. Error bars indicated in this figure represent mean and SD of MFI of T_reg-associated molecules expressing on CD4⁺ T cells in three independent experiments (n = 3, t test, P_A = 0.7015; P_B = 0.8555; P_C = 0.4659; P_D = 0.7642; P_E = 0.9505; NS, no significant difference).

Figure 7

IL-27 facilitates LPS-stimulated mature DC-mediated development of CD4⁺CD127⁺3G11⁺ T_reg subset ex vivo. Bone marrow-derived DCs were pulsed with MOG peptide and treated with IL-27 (20 ng/ml, 72 h) (DCs+IL-27) or LPS (DCs+LPS) (1 μg/ml, 24 h) or both LPS and IL-27 (DCs+LPS+IL-27). These DCs were then i.v. transferred into mice with EAE shown in Figure 4. Mice treated with PBS are control. Spleen cells were isolated and re-stimulated with MOG peptide (0.1 μM) and IL-2 (1 ng/ml) for 72 h. Cells were collected and CD4⁺CD25⁺FoxP3⁺GITR⁺ T_regs were gated. The frequency of CD127⁺3G11⁺ cells is demonstrated. T lymphocytes incubated with isotype control antibodies are isotype control. Error bars indicated in this figure represent mean and SD of frequency of CD4⁺CD127⁺3G11⁺ cells in three independent experiments [n = 3, t test, P_{(DC, DC+IL−27)} = 0.8682; P_{(DC+LPS, DC+LPS+IL−27)} = 0.0105].