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Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10-secreting Th1 cells.

Gabrysová L, Nicolson KS, Streeter HB, Verhagen J, Sabatos-Peyton CA, Morgan DJ, Wraith DC - J. Exp. Med. (2009)

Bottom Line: Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-gamma to the regulatory cytokine IL-10.IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology.These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of Bristol School of Medical Sciences, Bristol BS8 1TD, England, UK. Leona.Gabrysova@bristol.ac.uk

ABSTRACT
Regulation of the immune response to self- and foreign antigens is vitally important for limiting immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune conditions can be reinforced by tolerance induction with peptide epitopes, but the mechanism is not currently understood. Repetitive intranasal administration of soluble peptide induces peripheral tolerance in myelin basic protein (MBP)-specific TCR transgenic mice. This is characterized by the presence of anergic, interleukin (IL)-10-secreting CD4(+) T cells with regulatory function (IL-10 T reg cells). The differentiation pathway of peptide-induced IL-10 T reg cells was investigated. CD4(+) T cells became anergic after their second encounter with a high-affinity MBP peptide analogue. Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-gamma to the regulatory cytokine IL-10. Nevertheless, IL-10 T reg cells retained the capacity to produce IFN-gamma and concomitantly expressed T-bet, demonstrating their Th1 origin. IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.

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Expression of genes associated with differentiation of IL-10 T reg cells. (A–D) Tg4 Rag1−/− mice were treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y]. Total RNA was extracted from splenic CD4+ T cells positively selected from either untreated or peptide-treated mice at 2 h after each peptide treatment. The RNA was reverse transcribed and expression levels of the transcription factors t-bet, gata-3, egr-2, and the co-stimulatory molecule icos were determined by quantitative real-time PCR after normalization to β2-microglobulin expression. Results show the relative expression of the gene of interest from three accumulated experiments at each time point. (E) Enriched IL-10–secreting CD4+ T cells from Tg4 mice treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y] and in vitro–differentiated Th1 and Th2 cells were stained for surface Vβ8 and intracellular T-bet (black line) or isotype control (gray line). The depicted density plot of IL-10–enriched CD4+ T cells is gated on Vβ8+ T cells. The histogram of the IL-10+ cell fraction is gated on Vβ8+IL-10+ cells and Th1/Th2 cell histograms on Vβ8+ T cells. Data are representative of at least three individual experiments. (F) FACS-sorted IL-10+, IL-10+IFN-γ+, and IL-10+IFN-γ− Tg4 CD4+ T cells were analyzed for T-bet expression by Western blot, with Th1 and naive (CD4+CD62L+) cells used as positive and negative controls for T-bet detection, respectively. Data are representative of two individual experiments.
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fig4: Expression of genes associated with differentiation of IL-10 T reg cells. (A–D) Tg4 Rag1−/− mice were treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y]. Total RNA was extracted from splenic CD4+ T cells positively selected from either untreated or peptide-treated mice at 2 h after each peptide treatment. The RNA was reverse transcribed and expression levels of the transcription factors t-bet, gata-3, egr-2, and the co-stimulatory molecule icos were determined by quantitative real-time PCR after normalization to β2-microglobulin expression. Results show the relative expression of the gene of interest from three accumulated experiments at each time point. (E) Enriched IL-10–secreting CD4+ T cells from Tg4 mice treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y] and in vitro–differentiated Th1 and Th2 cells were stained for surface Vβ8 and intracellular T-bet (black line) or isotype control (gray line). The depicted density plot of IL-10–enriched CD4+ T cells is gated on Vβ8+ T cells. The histogram of the IL-10+ cell fraction is gated on Vβ8+IL-10+ cells and Th1/Th2 cell histograms on Vβ8+ T cells. Data are representative of at least three individual experiments. (F) FACS-sorted IL-10+, IL-10+IFN-γ+, and IL-10+IFN-γ− Tg4 CD4+ T cells were analyzed for T-bet expression by Western blot, with Th1 and naive (CD4+CD62L+) cells used as positive and negative controls for T-bet detection, respectively. Data are representative of two individual experiments.

Mentions: Further evidence in favor of the Th1 origin of peptide-induced IL-10 T reg cells was provided by the analysis of lineage-determining transcription factors. Changes in the relative mRNA expression of Th1 and Th2 master regulators t-bet and gata-3 by CD4+ T cells were monitored by quantitative real-time RT-PCR over the course of tolerization. As shown in Fig. 4 A, the relative expression of t-bet increased 25-fold after the first i.n. peptide treatment and reached a plateau of ∼100-fold after the second i.n. peptide treatment. Conversely, the relative expression of gata-3 decreased after the first i.n. peptide treatment in comparison to the level seen in untreated mice (Fig. 4 B). In addition to T-bet, a previous study revealed expression of the anergy-associated gene egr-2 and the co-stimulatory molecule icos in Tg4 CD4+ T cells from i.n. peptide-treated mice (Anderson et al., 2006). Expression of Egr-2 was strikingly up-regulated after the second i.n. peptide administration (Fig. 4 C), correlating with reduced proliferation (Fig. 2 A). Up-regulation of icos was delayed by one i.n. peptide treatment relative to egr-2 and T-bet (Fig. 4 D). Instead, icos expression appeared to be more closely associated with IL-10 secretion (Fig. 2, C and D). To assess T-bet expression by IL-10 T reg cells, we used a cytokine capture assay to enrich IL-10–secreting CD4+ T cells. The IL-10–secreting fraction comprised CD25high, CD69high to CD69intermediate, CD62Llow, and CD44high CD4+ T cells characteristic of activated, antigen-experienced CD4+ T cells (unpublished data). Essentially all IL-10–secreting CD4+ T cells expressed T-bet, as also found in in vitro–differentiated Th1, but not Th2, cells (Fig. 4 E). Because a significant proportion of IL-10–secreting CD4+ T cells from mice treated with 10 doses of i.n. peptide coproduced IFN-γ, CD4+ T cells from peptide-treated mice were sorted into IL-10+, IL-10+ IFN-γ+, and IL-10+ IFN-γ− CD4+ T cell fractions to reveal T-bet expression by each subset (Fig. 4 F). The Western blot results show T-bet protein expression in the sorted, IL-10–secreting T cell fraction as a whole, and importantly, in both the IL-10+ IFN-γ+ and IL-10+ IFN-γ− CD4+ T cell subpopulations. This clearly demonstrates that IL-10–secreting CD4+ T cells retain T-bet expression despite losing the ability to secrete IFN-γ, thus further confirming their Th1 origin.


Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10-secreting Th1 cells.

Gabrysová L, Nicolson KS, Streeter HB, Verhagen J, Sabatos-Peyton CA, Morgan DJ, Wraith DC - J. Exp. Med. (2009)

Expression of genes associated with differentiation of IL-10 T reg cells. (A–D) Tg4 Rag1−/− mice were treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y]. Total RNA was extracted from splenic CD4+ T cells positively selected from either untreated or peptide-treated mice at 2 h after each peptide treatment. The RNA was reverse transcribed and expression levels of the transcription factors t-bet, gata-3, egr-2, and the co-stimulatory molecule icos were determined by quantitative real-time PCR after normalization to β2-microglobulin expression. Results show the relative expression of the gene of interest from three accumulated experiments at each time point. (E) Enriched IL-10–secreting CD4+ T cells from Tg4 mice treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y] and in vitro–differentiated Th1 and Th2 cells were stained for surface Vβ8 and intracellular T-bet (black line) or isotype control (gray line). The depicted density plot of IL-10–enriched CD4+ T cells is gated on Vβ8+ T cells. The histogram of the IL-10+ cell fraction is gated on Vβ8+IL-10+ cells and Th1/Th2 cell histograms on Vβ8+ T cells. Data are representative of at least three individual experiments. (F) FACS-sorted IL-10+, IL-10+IFN-γ+, and IL-10+IFN-γ− Tg4 CD4+ T cells were analyzed for T-bet expression by Western blot, with Th1 and naive (CD4+CD62L+) cells used as positive and negative controls for T-bet detection, respectively. Data are representative of two individual experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2722173&req=5

fig4: Expression of genes associated with differentiation of IL-10 T reg cells. (A–D) Tg4 Rag1−/− mice were treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y]. Total RNA was extracted from splenic CD4+ T cells positively selected from either untreated or peptide-treated mice at 2 h after each peptide treatment. The RNA was reverse transcribed and expression levels of the transcription factors t-bet, gata-3, egr-2, and the co-stimulatory molecule icos were determined by quantitative real-time PCR after normalization to β2-microglobulin expression. Results show the relative expression of the gene of interest from three accumulated experiments at each time point. (E) Enriched IL-10–secreting CD4+ T cells from Tg4 mice treated with a minimum of 10 i.n. doses of MBP Ac1-9[4Y] and in vitro–differentiated Th1 and Th2 cells were stained for surface Vβ8 and intracellular T-bet (black line) or isotype control (gray line). The depicted density plot of IL-10–enriched CD4+ T cells is gated on Vβ8+ T cells. The histogram of the IL-10+ cell fraction is gated on Vβ8+IL-10+ cells and Th1/Th2 cell histograms on Vβ8+ T cells. Data are representative of at least three individual experiments. (F) FACS-sorted IL-10+, IL-10+IFN-γ+, and IL-10+IFN-γ− Tg4 CD4+ T cells were analyzed for T-bet expression by Western blot, with Th1 and naive (CD4+CD62L+) cells used as positive and negative controls for T-bet detection, respectively. Data are representative of two individual experiments.
Mentions: Further evidence in favor of the Th1 origin of peptide-induced IL-10 T reg cells was provided by the analysis of lineage-determining transcription factors. Changes in the relative mRNA expression of Th1 and Th2 master regulators t-bet and gata-3 by CD4+ T cells were monitored by quantitative real-time RT-PCR over the course of tolerization. As shown in Fig. 4 A, the relative expression of t-bet increased 25-fold after the first i.n. peptide treatment and reached a plateau of ∼100-fold after the second i.n. peptide treatment. Conversely, the relative expression of gata-3 decreased after the first i.n. peptide treatment in comparison to the level seen in untreated mice (Fig. 4 B). In addition to T-bet, a previous study revealed expression of the anergy-associated gene egr-2 and the co-stimulatory molecule icos in Tg4 CD4+ T cells from i.n. peptide-treated mice (Anderson et al., 2006). Expression of Egr-2 was strikingly up-regulated after the second i.n. peptide administration (Fig. 4 C), correlating with reduced proliferation (Fig. 2 A). Up-regulation of icos was delayed by one i.n. peptide treatment relative to egr-2 and T-bet (Fig. 4 D). Instead, icos expression appeared to be more closely associated with IL-10 secretion (Fig. 2, C and D). To assess T-bet expression by IL-10 T reg cells, we used a cytokine capture assay to enrich IL-10–secreting CD4+ T cells. The IL-10–secreting fraction comprised CD25high, CD69high to CD69intermediate, CD62Llow, and CD44high CD4+ T cells characteristic of activated, antigen-experienced CD4+ T cells (unpublished data). Essentially all IL-10–secreting CD4+ T cells expressed T-bet, as also found in in vitro–differentiated Th1, but not Th2, cells (Fig. 4 E). Because a significant proportion of IL-10–secreting CD4+ T cells from mice treated with 10 doses of i.n. peptide coproduced IFN-γ, CD4+ T cells from peptide-treated mice were sorted into IL-10+, IL-10+ IFN-γ+, and IL-10+ IFN-γ− CD4+ T cell fractions to reveal T-bet expression by each subset (Fig. 4 F). The Western blot results show T-bet protein expression in the sorted, IL-10–secreting T cell fraction as a whole, and importantly, in both the IL-10+ IFN-γ+ and IL-10+ IFN-γ− CD4+ T cell subpopulations. This clearly demonstrates that IL-10–secreting CD4+ T cells retain T-bet expression despite losing the ability to secrete IFN-γ, thus further confirming their Th1 origin.

Bottom Line: Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-gamma to the regulatory cytokine IL-10.IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology.These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, University of Bristol School of Medical Sciences, Bristol BS8 1TD, England, UK. Leona.Gabrysova@bristol.ac.uk

ABSTRACT
Regulation of the immune response to self- and foreign antigens is vitally important for limiting immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune conditions can be reinforced by tolerance induction with peptide epitopes, but the mechanism is not currently understood. Repetitive intranasal administration of soluble peptide induces peripheral tolerance in myelin basic protein (MBP)-specific TCR transgenic mice. This is characterized by the presence of anergic, interleukin (IL)-10-secreting CD4(+) T cells with regulatory function (IL-10 T reg cells). The differentiation pathway of peptide-induced IL-10 T reg cells was investigated. CD4(+) T cells became anergic after their second encounter with a high-affinity MBP peptide analogue. Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-gamma to the regulatory cytokine IL-10. Nevertheless, IL-10 T reg cells retained the capacity to produce IFN-gamma and concomitantly expressed T-bet, demonstrating their Th1 origin. IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.

Show MeSH
Related in: MedlinePlus