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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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RORγt+T-bet+ Th17 cells arise during CNS inflammation and T-bet-expressing, IL-23-induced Th17 cells are more pathogenic a, b, To induce CNS inflammation, we immunized Rorc(γt)-GfpTG or Rorc(γt)-GfpTG- control mice with MOG35–55 in CFA and CD4+ T cells isolated from the draining lymph nodes (dLN) or the CNS were analyzed by flow cytometry for IL-17, T-bet and GFP (RORγt) expression (upper panel). Lower panel shows isotype control staining for T-bet and fluorescence in transgene-negative littermates. A representative staining is depicted in a and pooled data of RORγt+ T-bet− and RORγt+ T-bet+ IL-17+CD4+ T cells are shown in b (n=7, *P<0.05). c, Naïve CD4+Vβ11+CD62L+CD44− were isolated by cell sorting from TCR(2D2) transgenic mice. The cells were activated with anti-CD3/anti-CD28, IL-6, IL-1β and anti-IFN-γ neutralizing antibodies with either TGF-β1 or IL-23 with anti-TGF-β neutralizing antibodies. The resultant cells were analyzed for IL-17 and IL-10 expression by intracellular staining and flow cytometry. d, Polarized cells (1×106) were adoptively transferred into Rag2−/− recipients and followed for signs of neurological disease. Data show mean ± s.e.m. of the EAE clinical score of 20 mice pooled from two independent experiments. e, f, CNS-infiltrating mononuclear cells were isolated and the total number of CD4+ T cells was determined in both groups (*P<0.01, e). The absolute numbers of CNS-infiltrating IL-17+ or IFN-γ+ CD4+ T cells of each group were assessed by intracellular cytokine staining (*P<0.05, **P<0.01, f).
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Figure 4: RORγt+T-bet+ Th17 cells arise during CNS inflammation and T-bet-expressing, IL-23-induced Th17 cells are more pathogenic a, b, To induce CNS inflammation, we immunized Rorc(γt)-GfpTG or Rorc(γt)-GfpTG- control mice with MOG35–55 in CFA and CD4+ T cells isolated from the draining lymph nodes (dLN) or the CNS were analyzed by flow cytometry for IL-17, T-bet and GFP (RORγt) expression (upper panel). Lower panel shows isotype control staining for T-bet and fluorescence in transgene-negative littermates. A representative staining is depicted in a and pooled data of RORγt+ T-bet− and RORγt+ T-bet+ IL-17+CD4+ T cells are shown in b (n=7, *P<0.05). c, Naïve CD4+Vβ11+CD62L+CD44− were isolated by cell sorting from TCR(2D2) transgenic mice. The cells were activated with anti-CD3/anti-CD28, IL-6, IL-1β and anti-IFN-γ neutralizing antibodies with either TGF-β1 or IL-23 with anti-TGF-β neutralizing antibodies. The resultant cells were analyzed for IL-17 and IL-10 expression by intracellular staining and flow cytometry. d, Polarized cells (1×106) were adoptively transferred into Rag2−/− recipients and followed for signs of neurological disease. Data show mean ± s.e.m. of the EAE clinical score of 20 mice pooled from two independent experiments. e, f, CNS-infiltrating mononuclear cells were isolated and the total number of CD4+ T cells was determined in both groups (*P<0.01, e). The absolute numbers of CNS-infiltrating IL-17+ or IFN-γ+ CD4+ T cells of each group were assessed by intracellular cytokine staining (*P<0.05, **P<0.01, f).

Mentions: It is well established that IL-23 is critical for the development of EAE, but T-bet is also critical, a finding that does not fit well with our current understanding of Th17 cell development15. The present findings suggest that Th17 cells, which arise in the absence TGF-β, would express RORγt and T-bet. To test if such cells are seen in vivo, we immunized Rorc(γt)-GfpTG-reporter mice16 with myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide. We found that approximately 25 to 60% of RORγt+ IL-17+ CD4+ cells in the CNS expressed T-bet (Fig. 4a, b). Furthermore, a significantly higher proportion of the RORγt, T-bet double positive Th17 cell population is found within the CNS compared with RORγt single positive Th17 cells (Fig. 4b).


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)

RORγt+T-bet+ Th17 cells arise during CNS inflammation and T-bet-expressing, IL-23-induced Th17 cells are more pathogenic a, b, To induce CNS inflammation, we immunized Rorc(γt)-GfpTG or Rorc(γt)-GfpTG- control mice with MOG35–55 in CFA and CD4+ T cells isolated from the draining lymph nodes (dLN) or the CNS were analyzed by flow cytometry for IL-17, T-bet and GFP (RORγt) expression (upper panel). Lower panel shows isotype control staining for T-bet and fluorescence in transgene-negative littermates. A representative staining is depicted in a and pooled data of RORγt+ T-bet− and RORγt+ T-bet+ IL-17+CD4+ T cells are shown in b (n=7, *P<0.05). c, Naïve CD4+Vβ11+CD62L+CD44− were isolated by cell sorting from TCR(2D2) transgenic mice. The cells were activated with anti-CD3/anti-CD28, IL-6, IL-1β and anti-IFN-γ neutralizing antibodies with either TGF-β1 or IL-23 with anti-TGF-β neutralizing antibodies. The resultant cells were analyzed for IL-17 and IL-10 expression by intracellular staining and flow cytometry. d, Polarized cells (1×106) were adoptively transferred into Rag2−/− recipients and followed for signs of neurological disease. Data show mean ± s.e.m. of the EAE clinical score of 20 mice pooled from two independent experiments. e, f, CNS-infiltrating mononuclear cells were isolated and the total number of CD4+ T cells was determined in both groups (*P<0.01, e). The absolute numbers of CNS-infiltrating IL-17+ or IFN-γ+ CD4+ T cells of each group were assessed by intracellular cytokine staining (*P<0.05, **P<0.01, f).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3108066&req=5

Figure 4: RORγt+T-bet+ Th17 cells arise during CNS inflammation and T-bet-expressing, IL-23-induced Th17 cells are more pathogenic a, b, To induce CNS inflammation, we immunized Rorc(γt)-GfpTG or Rorc(γt)-GfpTG- control mice with MOG35–55 in CFA and CD4+ T cells isolated from the draining lymph nodes (dLN) or the CNS were analyzed by flow cytometry for IL-17, T-bet and GFP (RORγt) expression (upper panel). Lower panel shows isotype control staining for T-bet and fluorescence in transgene-negative littermates. A representative staining is depicted in a and pooled data of RORγt+ T-bet− and RORγt+ T-bet+ IL-17+CD4+ T cells are shown in b (n=7, *P<0.05). c, Naïve CD4+Vβ11+CD62L+CD44− were isolated by cell sorting from TCR(2D2) transgenic mice. The cells were activated with anti-CD3/anti-CD28, IL-6, IL-1β and anti-IFN-γ neutralizing antibodies with either TGF-β1 or IL-23 with anti-TGF-β neutralizing antibodies. The resultant cells were analyzed for IL-17 and IL-10 expression by intracellular staining and flow cytometry. d, Polarized cells (1×106) were adoptively transferred into Rag2−/− recipients and followed for signs of neurological disease. Data show mean ± s.e.m. of the EAE clinical score of 20 mice pooled from two independent experiments. e, f, CNS-infiltrating mononuclear cells were isolated and the total number of CD4+ T cells was determined in both groups (*P<0.01, e). The absolute numbers of CNS-infiltrating IL-17+ or IFN-γ+ CD4+ T cells of each group were assessed by intracellular cytokine staining (*P<0.05, **P<0.01, f).
Mentions: It is well established that IL-23 is critical for the development of EAE, but T-bet is also critical, a finding that does not fit well with our current understanding of Th17 cell development15. The present findings suggest that Th17 cells, which arise in the absence TGF-β, would express RORγt and T-bet. To test if such cells are seen in vivo, we immunized Rorc(γt)-GfpTG-reporter mice16 with myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide. We found that approximately 25 to 60% of RORγt+ IL-17+ CD4+ cells in the CNS expressed T-bet (Fig. 4a, b). Furthermore, a significantly higher proportion of the RORγt, T-bet double positive Th17 cell population is found within the CNS compared with RORγt single positive Th17 cells (Fig. 4b).

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