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Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling.

Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O'Shea JJ - Nature (2010)

Bottom Line: Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β.T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model.These data indicate an alternative mode for T(H)17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. ghoreschik@mail.nih.gov

ABSTRACT
CD4(+) T-helper cells that selectively produce interleukin (IL)-17 (T(H)17), are critical for host defence and autoimmunity. Although crucial for T(H)17 cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been proposed to be the factors responsible for initiating specification. Here we show that T(H)17 differentiation can occur in the absence of TGF-β signalling. Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β. Epigenetic modification of the Il17a, Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed RORγt (encoded by Rorc) and T-bet. T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model. These data indicate an alternative mode for T(H)17 differentiation. Consistent with genetic data linking IL23R with autoimmunity, our findings re-emphasize the importance of IL-23 and therefore may have therapeutic implications.

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In vivo and in vitro differentiation of Th17 cells in the absence of TGF-β signalinga,b, Lamina propria cells were isolated from CD4dnTGFβRII and age-matched wild type (WT) mice (a) or Tgfbr1f/fCD4-Cre+ and Tgfbr1fl+CD4-Cre+ littermate controls (control) (b). Cells were stained for T cell markers and intracellular expression of IFN-γ, IL-17A, RORγt and FoxP3. Representative experiments are shown in left panels and pooled data are shown on the right (mean; error bars in b denote s.e.m., n=7). No significant differences in absolute numbers and proportions of IL-17A+CD4+ T cells were noted. *P<0.05. c–e, Naïve CD4+ T cells were isolated by cell sorting and activated in serum-free media with plate-bound anti-CD3/anti-CD28 for 4 days together with the indicated cytokines. Il17a mRNA expression was assessed by quantitative RT-PCR (c). IL-17A and IL-2 protein expression were analyzed by intracellular staining. Neutralizing anti-TGF-β antibodies prevented IL-6/IL-1β and TGF-β-dependent differentiation of Th17 cells, but not IL-23 and IL-6/IL-1β induced differentiation. Representative intracellular staining is depicted in panel d and pooled data from four individual experiments with mean values are shown in panel e. *P<0.05, **P<0.01.
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Figure 1: In vivo and in vitro differentiation of Th17 cells in the absence of TGF-β signalinga,b, Lamina propria cells were isolated from CD4dnTGFβRII and age-matched wild type (WT) mice (a) or Tgfbr1f/fCD4-Cre+ and Tgfbr1fl+CD4-Cre+ littermate controls (control) (b). Cells were stained for T cell markers and intracellular expression of IFN-γ, IL-17A, RORγt and FoxP3. Representative experiments are shown in left panels and pooled data are shown on the right (mean; error bars in b denote s.e.m., n=7). No significant differences in absolute numbers and proportions of IL-17A+CD4+ T cells were noted. *P<0.05. c–e, Naïve CD4+ T cells were isolated by cell sorting and activated in serum-free media with plate-bound anti-CD3/anti-CD28 for 4 days together with the indicated cytokines. Il17a mRNA expression was assessed by quantitative RT-PCR (c). IL-17A and IL-2 protein expression were analyzed by intracellular staining. Neutralizing anti-TGF-β antibodies prevented IL-6/IL-1β and TGF-β-dependent differentiation of Th17 cells, but not IL-23 and IL-6/IL-1β induced differentiation. Representative intracellular staining is depicted in panel d and pooled data from four individual experiments with mean values are shown in panel e. *P<0.05, **P<0.01.

Mentions: TGF-β1 is important for murine Th17 differentiation7,8 and accordingly, transgenic expression of a mutant TGF-β subunit receptor II (CD4dnTGFβRII) in T cells interferes with the generation of Th17 cells in the setting of EAE11. We revisited this issue by assessing whether Th17 cells were present in the intestinal lamina propria of these mice. As expected, increased proportions and absolute numbers of IFN-γ-producing CD4+ T cells were present (Supplementary Fig. 1a). Of note, IL-17-producing CD4+ cells were also present; the proportions were reduced, but there were no differences in the absolute numbers between wild type and CD4dnTGFβRII mice (Fig. 1a). We confirmed this finding using mice that lack TGF-β receptor subunit I in their T cells (Tgfbr1f/fCD4-Cre+ mice)12 (Fig. 1b). Rorγt-expressing CD4+ lamina propria T cells were also present in Tgfbr1f/fCD4-Cre+ mice, whereas Foxp3+CD4+ T cells were dramatically reduced (Fig. 1b). Collectively, these data argue that in vivo Th17 differentiation can occur in the absence of TGF-β signaling.


Generation of pathogenic T(H)17 cells in the absence of TGF-β signalling.

Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O'Shea JJ - Nature (2010)

In vivo and in vitro differentiation of Th17 cells in the absence of TGF-β signalinga,b, Lamina propria cells were isolated from CD4dnTGFβRII and age-matched wild type (WT) mice (a) or Tgfbr1f/fCD4-Cre+ and Tgfbr1fl+CD4-Cre+ littermate controls (control) (b). Cells were stained for T cell markers and intracellular expression of IFN-γ, IL-17A, RORγt and FoxP3. Representative experiments are shown in left panels and pooled data are shown on the right (mean; error bars in b denote s.e.m., n=7). No significant differences in absolute numbers and proportions of IL-17A+CD4+ T cells were noted. *P<0.05. c–e, Naïve CD4+ T cells were isolated by cell sorting and activated in serum-free media with plate-bound anti-CD3/anti-CD28 for 4 days together with the indicated cytokines. Il17a mRNA expression was assessed by quantitative RT-PCR (c). IL-17A and IL-2 protein expression were analyzed by intracellular staining. Neutralizing anti-TGF-β antibodies prevented IL-6/IL-1β and TGF-β-dependent differentiation of Th17 cells, but not IL-23 and IL-6/IL-1β induced differentiation. Representative intracellular staining is depicted in panel d and pooled data from four individual experiments with mean values are shown in panel e. *P<0.05, **P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108066&req=5

Figure 1: In vivo and in vitro differentiation of Th17 cells in the absence of TGF-β signalinga,b, Lamina propria cells were isolated from CD4dnTGFβRII and age-matched wild type (WT) mice (a) or Tgfbr1f/fCD4-Cre+ and Tgfbr1fl+CD4-Cre+ littermate controls (control) (b). Cells were stained for T cell markers and intracellular expression of IFN-γ, IL-17A, RORγt and FoxP3. Representative experiments are shown in left panels and pooled data are shown on the right (mean; error bars in b denote s.e.m., n=7). No significant differences in absolute numbers and proportions of IL-17A+CD4+ T cells were noted. *P<0.05. c–e, Naïve CD4+ T cells were isolated by cell sorting and activated in serum-free media with plate-bound anti-CD3/anti-CD28 for 4 days together with the indicated cytokines. Il17a mRNA expression was assessed by quantitative RT-PCR (c). IL-17A and IL-2 protein expression were analyzed by intracellular staining. Neutralizing anti-TGF-β antibodies prevented IL-6/IL-1β and TGF-β-dependent differentiation of Th17 cells, but not IL-23 and IL-6/IL-1β induced differentiation. Representative intracellular staining is depicted in panel d and pooled data from four individual experiments with mean values are shown in panel e. *P<0.05, **P<0.01.
Mentions: TGF-β1 is important for murine Th17 differentiation7,8 and accordingly, transgenic expression of a mutant TGF-β subunit receptor II (CD4dnTGFβRII) in T cells interferes with the generation of Th17 cells in the setting of EAE11. We revisited this issue by assessing whether Th17 cells were present in the intestinal lamina propria of these mice. As expected, increased proportions and absolute numbers of IFN-γ-producing CD4+ T cells were present (Supplementary Fig. 1a). Of note, IL-17-producing CD4+ cells were also present; the proportions were reduced, but there were no differences in the absolute numbers between wild type and CD4dnTGFβRII mice (Fig. 1a). We confirmed this finding using mice that lack TGF-β receptor subunit I in their T cells (Tgfbr1f/fCD4-Cre+ mice)12 (Fig. 1b). Rorγt-expressing CD4+ lamina propria T cells were also present in Tgfbr1f/fCD4-Cre+ mice, whereas Foxp3+CD4+ T cells were dramatically reduced (Fig. 1b). Collectively, these data argue that in vivo Th17 differentiation can occur in the absence of TGF-β signaling.

Bottom Line: Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β.T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model.These data indicate an alternative mode for T(H)17 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology and Inflammation Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. ghoreschik@mail.nih.gov

ABSTRACT
CD4(+) T-helper cells that selectively produce interleukin (IL)-17 (T(H)17), are critical for host defence and autoimmunity. Although crucial for T(H)17 cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been proposed to be the factors responsible for initiating specification. Here we show that T(H)17 differentiation can occur in the absence of TGF-β signalling. Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β. Epigenetic modification of the Il17a, Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed RORγt (encoded by Rorc) and T-bet. T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model. These data indicate an alternative mode for T(H)17 differentiation. Consistent with genetic data linking IL23R with autoimmunity, our findings re-emphasize the importance of IL-23 and therefore may have therapeutic implications.

Show MeSH
Related in: MedlinePlus