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Tolerogenic splenic IDO (+) dendritic cells from the mice treated with induced-Treg cells suppress collagen-induced arthritis.

Yang J, Yang Y, Fan H, Zou H - J Immunol Res (2014)

Bottom Line: In this study, we found that after treatment with iTregs, splenic CD11c(+)DCs, termed "DCiTreg," expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activity in vitro.Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner.Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Huashan Hospital, Fudan University, Shanghai 200040, China ; Blood Engineering Laboratory, Shanghai Blood Center, Shanghai 200051, China.

ABSTRACT
TGF-β-induced regulatory T cells (iTregs) retain Foxp3 expression and immune-suppressive activity in collagen-induced arthritis (CIA). However, the mechanisms whereby transferred iTregs suppress immune responses, particularly the interplay between iTregs and dendritic cells (DCs) in vivo, remain incompletely understood. In this study, we found that after treatment with iTregs, splenic CD11c(+)DCs, termed "DCiTreg," expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activity in vitro. After reinfusion with DCiTreg, marked antiarthritic activity improved clinical scores and histological end-points were observed. The serological levels of inflammatory cytokines and anti-CII antibodies were low and TGF-β production was high in the DCiTreg-treated group. DCiTreg also induced new iTregs in vivo. Moreover, the inhibitory activity of DCiTreg on CIA was lost following pretreatment with the inhibitor of indoleamine 2,3-dioxygenase (IDO). Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner. Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCs in vivo.

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The conversion of Foxp3+Tregs from CD4+CD25−T cells through coculture with isolated splenic CD11b+IDO+DCs. (a) CD4+CD25−T cells (105) stimulated with mDCs (2 × 104) were cocultured for 4 days with isolated splenic CD11b+DCs (104) with or without 1-MT pretreatment. The cells were surface-stained with anti-CD4 mAbs, followed by intracellular staining with anti-Foxp3 mAbs to determine the frequency of CD4+Foxp3+Tregs. The dates are presented using a dot plot, expressed as the % positive cells. The results are representative of three experiments showing similar results. (b) After cocultivation for 4 days, as described in (a), CD11b+DC-induced CD4+CD25+T cells were isolated and used as inhibitors at different doses to suppress the proliferation of new CD4+CD25−T cells in response to stimulation with anti-CD3/28 mAbs. The suppressive activity of freshly isolated CD4+CD25+T cells was also examined as a control. The proliferative responses were measured after 4 days. The data are representative of three experiments with similar results, reported as the means ± SEM. #P > 0.05 compared with the indicated groups using unpaired t-tests. (c) CD4+T cells from the spleens of CIA mice treated with CD11b+DC with or without 1-MT on the third week after the onset of arthritis were collected and intracellularly stained with anti-Foxp3 and anti-IL-17A mAbs. FACS was used to measure the percent frequency of positively stained cells, and the frequencies of Treg/Th17 cells are expressed as the means ± SEM of four independent experiments. #P > 0.05, *P < 0.05 compared with the indicated groups using unpaired t-tests.
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fig5: The conversion of Foxp3+Tregs from CD4+CD25−T cells through coculture with isolated splenic CD11b+IDO+DCs. (a) CD4+CD25−T cells (105) stimulated with mDCs (2 × 104) were cocultured for 4 days with isolated splenic CD11b+DCs (104) with or without 1-MT pretreatment. The cells were surface-stained with anti-CD4 mAbs, followed by intracellular staining with anti-Foxp3 mAbs to determine the frequency of CD4+Foxp3+Tregs. The dates are presented using a dot plot, expressed as the % positive cells. The results are representative of three experiments showing similar results. (b) After cocultivation for 4 days, as described in (a), CD11b+DC-induced CD4+CD25+T cells were isolated and used as inhibitors at different doses to suppress the proliferation of new CD4+CD25−T cells in response to stimulation with anti-CD3/28 mAbs. The suppressive activity of freshly isolated CD4+CD25+T cells was also examined as a control. The proliferative responses were measured after 4 days. The data are representative of three experiments with similar results, reported as the means ± SEM. #P > 0.05 compared with the indicated groups using unpaired t-tests. (c) CD4+T cells from the spleens of CIA mice treated with CD11b+DC with or without 1-MT on the third week after the onset of arthritis were collected and intracellularly stained with anti-Foxp3 and anti-IL-17A mAbs. FACS was used to measure the percent frequency of positively stained cells, and the frequencies of Treg/Th17 cells are expressed as the means ± SEM of four independent experiments. #P > 0.05, *P < 0.05 compared with the indicated groups using unpaired t-tests.

Mentions: To determine the effects of these cells in vitro, splenic CD11b+DCs isolated from iTreg-treated CIA mice were added to the CD4+CD25−T cell proliferation system, and after 4 days, the frequency of Foxp3+Tregs was analyzed via FACS. As indicated in Figure 5(a), the addition of CD11b+DCs markedly impaired Foxp3−T cell proliferation. Oppositely, the percentage of Foxp3 in effector cells was increased, suggesting that CD11b+DCs promoted the conversion of naïve CD4+CD25−Foxp3−T cells into adoptive CD4+Foxp3+Tregs. Furthermore, these DCs lost inhibitory activity when pretreated with 1-MT.


Tolerogenic splenic IDO (+) dendritic cells from the mice treated with induced-Treg cells suppress collagen-induced arthritis.

Yang J, Yang Y, Fan H, Zou H - J Immunol Res (2014)

The conversion of Foxp3+Tregs from CD4+CD25−T cells through coculture with isolated splenic CD11b+IDO+DCs. (a) CD4+CD25−T cells (105) stimulated with mDCs (2 × 104) were cocultured for 4 days with isolated splenic CD11b+DCs (104) with or without 1-MT pretreatment. The cells were surface-stained with anti-CD4 mAbs, followed by intracellular staining with anti-Foxp3 mAbs to determine the frequency of CD4+Foxp3+Tregs. The dates are presented using a dot plot, expressed as the % positive cells. The results are representative of three experiments showing similar results. (b) After cocultivation for 4 days, as described in (a), CD11b+DC-induced CD4+CD25+T cells were isolated and used as inhibitors at different doses to suppress the proliferation of new CD4+CD25−T cells in response to stimulation with anti-CD3/28 mAbs. The suppressive activity of freshly isolated CD4+CD25+T cells was also examined as a control. The proliferative responses were measured after 4 days. The data are representative of three experiments with similar results, reported as the means ± SEM. #P > 0.05 compared with the indicated groups using unpaired t-tests. (c) CD4+T cells from the spleens of CIA mice treated with CD11b+DC with or without 1-MT on the third week after the onset of arthritis were collected and intracellularly stained with anti-Foxp3 and anti-IL-17A mAbs. FACS was used to measure the percent frequency of positively stained cells, and the frequencies of Treg/Th17 cells are expressed as the means ± SEM of four independent experiments. #P > 0.05, *P < 0.05 compared with the indicated groups using unpaired t-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: The conversion of Foxp3+Tregs from CD4+CD25−T cells through coculture with isolated splenic CD11b+IDO+DCs. (a) CD4+CD25−T cells (105) stimulated with mDCs (2 × 104) were cocultured for 4 days with isolated splenic CD11b+DCs (104) with or without 1-MT pretreatment. The cells were surface-stained with anti-CD4 mAbs, followed by intracellular staining with anti-Foxp3 mAbs to determine the frequency of CD4+Foxp3+Tregs. The dates are presented using a dot plot, expressed as the % positive cells. The results are representative of three experiments showing similar results. (b) After cocultivation for 4 days, as described in (a), CD11b+DC-induced CD4+CD25+T cells were isolated and used as inhibitors at different doses to suppress the proliferation of new CD4+CD25−T cells in response to stimulation with anti-CD3/28 mAbs. The suppressive activity of freshly isolated CD4+CD25+T cells was also examined as a control. The proliferative responses were measured after 4 days. The data are representative of three experiments with similar results, reported as the means ± SEM. #P > 0.05 compared with the indicated groups using unpaired t-tests. (c) CD4+T cells from the spleens of CIA mice treated with CD11b+DC with or without 1-MT on the third week after the onset of arthritis were collected and intracellularly stained with anti-Foxp3 and anti-IL-17A mAbs. FACS was used to measure the percent frequency of positively stained cells, and the frequencies of Treg/Th17 cells are expressed as the means ± SEM of four independent experiments. #P > 0.05, *P < 0.05 compared with the indicated groups using unpaired t-tests.
Mentions: To determine the effects of these cells in vitro, splenic CD11b+DCs isolated from iTreg-treated CIA mice were added to the CD4+CD25−T cell proliferation system, and after 4 days, the frequency of Foxp3+Tregs was analyzed via FACS. As indicated in Figure 5(a), the addition of CD11b+DCs markedly impaired Foxp3−T cell proliferation. Oppositely, the percentage of Foxp3 in effector cells was increased, suggesting that CD11b+DCs promoted the conversion of naïve CD4+CD25−Foxp3−T cells into adoptive CD4+Foxp3+Tregs. Furthermore, these DCs lost inhibitory activity when pretreated with 1-MT.

Bottom Line: In this study, we found that after treatment with iTregs, splenic CD11c(+)DCs, termed "DCiTreg," expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activity in vitro.Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner.Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Huashan Hospital, Fudan University, Shanghai 200040, China ; Blood Engineering Laboratory, Shanghai Blood Center, Shanghai 200051, China.

ABSTRACT
TGF-β-induced regulatory T cells (iTregs) retain Foxp3 expression and immune-suppressive activity in collagen-induced arthritis (CIA). However, the mechanisms whereby transferred iTregs suppress immune responses, particularly the interplay between iTregs and dendritic cells (DCs) in vivo, remain incompletely understood. In this study, we found that after treatment with iTregs, splenic CD11c(+)DCs, termed "DCiTreg," expressed tolerogenic phenotypes, secreted high levels of IL-10, TGF-β, and IDO, and showed potent immunosuppressive activity in vitro. After reinfusion with DCiTreg, marked antiarthritic activity improved clinical scores and histological end-points were observed. The serological levels of inflammatory cytokines and anti-CII antibodies were low and TGF-β production was high in the DCiTreg-treated group. DCiTreg also induced new iTregs in vivo. Moreover, the inhibitory activity of DCiTreg on CIA was lost following pretreatment with the inhibitor of indoleamine 2,3-dioxygenase (IDO). Collectively, these findings suggest that transferred iTregs could induce tolerogenic characteristics in splenic DCs and these cells could effectively dampen CIA in an IDO-dependent manner. Thus, the potential therapeutic effects of iTregs in CIA are likely maintained through the generation of tolerogenic DCs in vivo.

Show MeSH
Related in: MedlinePlus