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Keywords:

  • Cytokine memory;
  • IL-17;
  • T-cell differentiation;
  • Th1/Th17 cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Th1 and Th17 cells are distinct lineages of effector/memory cells, imprinted for re-expression of IFN-γ and IL-17, by upregulated expression of T-bet and retinoic acid-related orphan receptor γt (RORγt), respectively. Apparently, Th1 and Th17 cells share tasks in the control of inflammatory immune responses. Th cells coexpressing IFN-γ and IL-17 have been observed in vivo, but it remained elusive, how these cells had been generated and whether they represent a distinct lineage of Th differentiation. It has been shown that ex vivo isolated Th1 and Th17 cells are not interconvertable by TGF-β/IL-6 and IL-12, respectively. Here, we show that ex vivo isolated Th17 cells can be converted into Th1/Th17 cells by combined IFN-γ and IL-12 signaling. IFN-γ is required to upregulate expression of the IL-12Rβ2 chain, and IL-12 for Th1 polarization. These Th1/Th17 cells stably coexpress RORγt and T-bet at the single-cell level. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, which combine the pro-inflammatory potential of Th1 and Th17 cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Th1 cells, with a memory for IFN-γ expression and determined by the master transcription factor T-bet, are considered to be essential for protection against intracellular pathogens, and had been viewed as the major pathogenic drivers of chronic autoimmune inflammation, e.g. EAE 1–3, uveitis 4 or colitis 5. Recently, Th17 cells, with a memory for expression of IL-17 and determined by the transcription factor retinoic acid related orphan receptor γt (RORγt), have been identified as another pathogenic Th-cell lineage driving pathogenesis in these autoimmune models 6, 7. Th17 cells contribute to inflammation through the recruitment of neutrophils and the induction of secretion of pro-inflammatory mediators such as IL-6, IL-8, TNF-α, IL-1β, CXCL1, CXCL10 and matrix metalloproteinases from tissue cells (reviewed in 8). Th1 cells contribute to inflammation by activation of macrophages 9. The concerted action of IFN-γ and IL-17 has been shown to be essential in the effective induction and maintenance of autoimmunity 10, 11, e.g. Th1 cells being required for the recruitment of Th17 cells into the central nervous system in EAE.

In inflamed tissue of autoimmune patients, Th cells coexpressing IFN-γ and IL-17 have been identified 12–14. However, the genesis and stability of such Th cells had remained enigmatic. In vitro, the differentiation of naïve Th cells into Th1 or Th17 cells is mutually exclusive, using the polarizing signals identified so far. TGF-β blocks the induction of expression of T-bet and IFN-γ 15. IFN-γ blocks the differentiation of naïve Th cells into Th17 cells 16. We have previously shown that murine Th17 cells isolated ex vivo cannot be converted into Th1 cells by IL-12, or into Th2 cells by IL-4 17, supporting the notion that Th17 cells are a distinct, stable and independent lineage of Th memory/effector differentiation. It has, however, been demonstrated that human Th-cell clones coexpressing IL-17 and IL-4 can be generated by stimulation of Th17 containing CCR6+CD161+ Th cells with IL-4 18.

Here, we demonstrate that Th17 cells can be induced to develop further, into Th1/Th17 cells, by the combined action of IFN-γ and IL-12. We show that ex vivo isolated Th17 cells lack IL-12Rβ2 expression and are not responsive to IL-12 alone. However, the expression of IL-12Rβ2 can be induced with IFN-γ, restoring IL-12 responsiveness. Stimulation of ex vivo isolated Th17 cells with IFN-γ and IL-12 results in stable induction of T-bet and functional imprinting of the Ifnγ gene for re-expression. Expression of RORγt is maintained, as well as functional imprinting of the Il17 gene for re-expression. Individual Th1/Th17 cells stably coexpress T-bet and RORγt. Our results suggest a molecular pathway for the generation of Th1/Th17 cells in vivo, and define Th1/Th17 cells as a functionally distinct Th population, combining the pro-inflammatory potential of Th1 and Th17 cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

In vivo, Th17 cells do not express IL-12Rβ2 and do not respond to IL-12

Although IL-17-expressing cells isolated from cultures stimulated in vitro respond to subsequent stimulation with IL-12 with gain of IFN-γ expression and loss of IL-17 expression (Fig. 1A, upper panel), IL-17 expressing cells directly isolated ex vivo maintained IL-17 expression and could not be induced to express IFN-γ in the presence of IL-12 (Fig. 1A, lower panel) 17, 19. To identify the molecular mechanism of refraction of in vivo generated Th17 cells to conversion by IL-12, we here compared the expression of genes relevant for IL-12 signaling by Th17 cells generated in vitro, and CD4+ T cells isolated directly ex vivo according to secretion of IL-17, using the cytometric cytokine secretion assay for IL-17, as described earlier 17. Expression levels of the lineage determining transcription factors for Th1 and Th17 cells, T-bet and RORγt, respectively, the IFN-γ receptor 2 (IFN-γR2) and the inducible IL-12Rβ2 chain were determined by quantitative PCR (Fig. 1B). Expression of the transcription factor RORγt in ex vivo isolated IL-17+ T cells was threefold higher as compared with in vitro-generated IL-17+ Th cells. Increased T-bet mRNA levels (threefold) were detected in the ex vivo-isolated Th17 cells compared with in vitro-generated Th17 cells (Fig. 1B). Directly, ex vivo isolated IL-17+ Th cells expressed fivefold less IL-12Rβ2 transcripts than in vitro-generated Th17 cells (Fig. 1B). Allowing the cells to rest did not significantly increase IL-12Rβ2 expression of ex vivo isolated Th17 cells (data not shown), suggesting that Il12rβ2 chain expression of Th17 cells is downregulated constitutively in vivo. Ex vivo isolated IL-17+ Th cells stimulated with IL-12 did not respond by STAT4 phosphorylation, demonstrating the absence of a functional IL-12 receptor on these cells (Fig. 1C). On the contrary, stimulation of ex vivo isolated IFN-γ+ Th cells resulted in STAT4 phosphorylation in more than 50% of the cells (Supporting Information Fig. 2). In vitro-generated Th17 cells responded to IL-12, by phosphorylation of STAT4 in all cells (Fig. 1C), and expression of IFN-γ by more than 40% of the cells (17 and data not shown).

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Figure 1. Ex vivo isolated Th17 cells express a functional IFN-γ receptor but lack a functional IL-12 receptor. (A) CD4+ T cells from spleen and lymph nodes from ex-breeder BALB/c mice and Th17 cells, generated in vitro by stimulating naïve CD4+CD62Lhigh cells with TGF-β, IL-6, IL-23, anti-IL-4 and anti-IFN-γ for 5 days, were stimulated for 5 h with PMA/ionomycin and stained intracellularly for IL-17 and IFN-γ (gated on CD4+ lymphocytes, Supporting Information Fig. 1A and B). IL-17-producing Th cells were isolated with an IL-17 secretion assay, stimulated with anti-CD3/anti-CD28 and cultured in the presence of IL-12 and absence of IL-4 and IFN-γ. Data are representative of five independent experiments. (B) mRNA expression of RORγt, IL-12Rβ2, IFN-γR2 and T-bet was determined in IL-17-secreting cells isolated directly ex vivo (ev) and from in vitro (iv)-induced Th17 cells and normalized to the housekeeping gene hypoxanthine guanine phosphoribosyl transferase. Data show mean±SD of three independent experiments. (C) IL-17-secreting cells isolated ex vivo and generated in vitro were rested for 2 days in the absence of IL-4, IFN-γ and IL-12. The cells were then stimulated for 30 min with IL-12 prior to intracellular staining of pSTAT4 or 15 min with IFN-γ for staining of pSTAT1. Scatter characteristics are shown in Supporting Information Fig. 1C. Cells incubated with culture medium alone served as negative control. Data are representative of three independent experiments.

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To confirm that downregulation of IL-12Rβ2 chain expression is not a transient consequence of TCR activation 20, 21, we directly isolated splenic Th cells based on the surface expression of IL-12Rβ2 (Fig. 2A, gating for viable CD4+ Th cells shown in Supporting Information Fig. 1D) and then quantified cells expressing intracellular IL-17 upon reactivation, among the IL-12Rβ2+ and IL-12β2 Th cells. Within the IL-12Rβ2CD4+ T cells, we could detect 0.66% IL-17-expressing cells, whereas 0.18% of the IL-12Rβ2+ Th cells expressed IL-17 (Fig. 2B). In terms of absolute cell numbers, we could detect 4×104±6×103 IL-17-expressing cells per murine spleen, which did not coexpress IL-12Rβ2, and 2×103±1×103 Th cells coexpressing IL-17 and IL-12Rβ2 (Fig. 2C). This means that in vivo 95% of murine Th17 cells found in the spleen do not express a functional IL-12 receptor.

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Figure 2. IL-17-expressing Th cells are enriched within the IL-12Rβ2low subset. (A) CD4+ T cells from spleen and lymph nodes from ex-breeder BALB/c mice were isolated with CD4 MACS beads. The cells were stained for IL-12Rβ2 and IL-12Rβ2high and IL-12Rβ2low CD4+ Th cells were isolated by FACS sorting. Data are representative of three independent experiments. (B) The sorted IL-12Rβ2high and IL-12Rβ2low CD4+ Th cells were stimulated with PMA/ionomycin for 4 h for recall expression of cytokines. The percentage of IL-17-producing cells per spleen was determined by intracellular cytokine staining. (C) Absolute cell numbers of IL-17-producing cells were determined with a MACSQuant Analyzer. Data show mean±SD of three independent experiments.

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IFN-γ induces IL-12Rβ2 expression by Th17 cells and restores responsiveness to IL-12

The Ifnγr2 gene was three- to four-fold higher expressed by Th17 cells, both in vivo and in vitro generated, than in Th1 cells (data not shown). This is in accordance with the previous reports demonstrating upregulation of Ifnγr expression by IL-6 22 and downregulation of Ifnγr expression in Th1 cells in vitro23. In in vitro-generated and directly ex vivo isolated Th17 cells, the IFN-γ receptor is functional. STAT1 phosphorylation was induced upon stimulation with IFN-γ (Fig. 1C), whereas Th1 cells did not respond to IFN-γ (data not shown). Interestingly, IFN-γ-induced activation of STAT1 was not sufficient to induce significant expression of Ifnγ (Fig. 3A), unlike shown previously in naïve Th cells 20, 24. Stimulation with IFN-γ did not change the expression level of RORγt but resulted in a detectable increase of T-bet expression as assessed by intracellular immunofluorescence (Fig. 3B). However, in ex vivo isolated Th17 cells, IFN-γ induced the expression of Il12Rβ2 (Fig. 3C) and functionally restored responsiveness to IL-12 (Fig. 3D) as has been shown for Th1 and Th2 cells before 25. When prestimulated with IFN-γ, IL-12 induced phosphorylation of STAT4 in more than 50% of the cells (Fig. 3D). IL-12, as previously shown 17, did not lead to significant induction of IFN-γ expression (Fig. 3A), nor upregulation of T-bet expression or changes in RORγt expression (Fig. 3B), upregulation of Il12Rβ2 (Fig. 3C) or responsiveness to IL-12 (Fig. 3D).

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Figure 3. IL-12Rβ2 expression is induced in ex vivo isolated Th17 cells by IFN-γ. (A) Ex vivo isolated IL-17+ cells were cultured in the absence of IFN-γ and IL-12 or in the presence of IFN-γ or IL-12 only for 5 days and stained intracellularly for IFN-γ and IL-17 expression. IL-4 was blocked under all conditions. Data are representative of five independent experiments. (B) mRNA was isolated from directly isolated IL-17+ Th cells, IL-17+ Th cells cultured in the absence of cytokines, in the presence of IFN-γ only or IL-12 only. IL-12Rβ2 expression was determined by real-time PCR. Experiment is representative of two independent experiments. (C) Intracellular RORγt and T-bet expression was measured after 5 days cell culture under the indicated conditions. Data are representative of three independent experiments. (D) pSTAT4 in response to IL-12 was measured by intracellular staining in IL-17+ Th cells cultured under the indicated conditions. Data are representative of three independent experiments.

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IFN-γ and IL-12 synergize to induce Th1/Th17 cells

Following restoration of responsiveness to IL-12 by prestimulation with IFN-γ, expression of the Ifnγ gene could be induced by IL-12 stimulation (Fig. 4A). All of the cells uniformly had upregulated T-bet expression, as determined by intracellular immunofluorescence, and 50% expressed intracellular IFN-γ. Although inducing expression of T-bet and IFN-γ in ex vivo isolated Th17 cells, combined IFN-γ and IL-12 signaling did not suppress expression of RORγt. All cells uniformly continued to express RORγt (Fig. 4B and C). Upon restimulation, they also re-expressed Il17. The frequencies of IL-17-producing cells dropped from 75% (±10%) to 38% (±6%). In total, 20% of the cells expressed only IL-17, 20% IL-17 and IFN-γ, and 30% only IFN-γ (Fig. 4A). The frequency of cells coexpressing IL-17 and IFN-γ observed (obs: 20%) corresponds to the frequency one would expect from the random coincidence of coexpression (exp: 20%), suggesting that the expression of IL-17 and IFN-γ is neither positively nor negatively correlated 26. Repeated stimulation of ex vivo isolated Th17 cells from IFN-γR2-deficient mice with IL-12 alone did not lead to a significant induction of IFN-γ in these cells (Supporting Information Fig. 3). We also exclude the selective outgrowth of pre-existing IFN-γ expressing cells as stimulation with IL-12 of isolated IL-17-expressing cells depleted of IFN-γ+ cells also resulted in the induction of cells coexpressing IFN-γ and IL-17 (Supporting Information Fig. 4).

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Figure 4. IFN-γ and IL-12 synergize to induce Th1/Th17 cells. (A) Directly ex vivo isolated IL-17+ CD4 T cells were cultured for 5 days in the presence of IFN-γ and then restimulated in the presence of either IFN-γ or IL-12 for another 5 days. Shown is the intracellular staining for IFN-γ and IL-17 expression after each culture. Data are representative of four independent experiments. (B and C) RORγt and T-bet were stained intracellularly following the second culture. Data are representative of four independent experiments.

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Th1/Th17 cells coexpress T-bet and RORγt

To analyze the stability of Th1/Th17 cells generated in vivo, we have immunized wild-type mice with peptide derived from myelin oligodendrocyte glycoprotein35–55 in complete Freund's adjuvant. Before onset of any clinical symptoms of EAE (Fig. 5A), we have assessed the expression of the cytokines IFN-γ and IL-17 in splenic CD4+ Th cells at day 7 post-immunization. Following restimulation with PMA and ionomycin, 5.56% of the splenic CD4+ Th cells expressed only IFN-γ, whereas 2.58% expressed only IL-17. In total, 0.62% of all splenic CD4+ Th cells coexpressed IL-17 and IFN-γ (Fig. 5A). On the contrary, in age-matched, unimmunized control mice, no IL-17-expressing or IFN-γ/IL-17 coexpressing CD4+ Th cells could be detected (Fig. 5A). Using an IFN-γ/IL-17 double secretion assay, we have isolated from spleen Th cells expressing either IFN-γ only, IL-17 only or coexpressing IL-17 and IFN-γ (Fig. 5B). Th cells expressing only IFN-γ uniformly expressed elevated T-bet levels (Δgeo mean of 1152 compared with negative control), whereas RORγt expression was low but detectable. IL-17 only-expressing Th cells expressed high RORγt levels (Δgeo mean of 3387) and detectable levels of T-bet (Δgeo mean of 518), similar to the ex vivo isolated Th17 cells shown in Fig. 1. Th cells expressing both IL-17 and IFN-γ uniformly expressed elevated levels of RORγt and T-bet (Δgeo mean of 2368 and 1138, respectively) (Fig. 5C). To confirm the intracellular staining, the mRNA expression of RORγt and T-bet in the three isolated subsets was quantified. T-bet and RORγt expression in the IL-17+IFN-γ+ Th1/Th17 cells was at similar levels as T-bet and RORγt levels in IFN-γ single-positive and IL-17 single-positive Th cells, respectively (Fig. 5D). Analysis of the expression levels of RORα, IL-12Rβ2, IFN-γR2 and L-23R also confirmed that Th1/Th17 cells share the properties of IL-17+ and IFN-γ+ Th cells (Supporting Information Fig. 5). The directly ex vivo isolated Th1/Th17 cells were then cultured without the addition of any blocking antibodies and exogenous cytokines (neutral) for 5 or 10 days. Upon restimulation with PMA/ionomycin, 23±1% of the cells coexpressed IL-17 and IFN-γ, 55±4% only IFN-γ and 12±3% only IL-17 after 5 days (Fig. 5E). The relative distribution of cytokine producers was maintained at similar levels after day 10 (12±6% coexpressing IL-17 and IFN-γ, 59±14% only IFN-γ and 14±6% only IL-17) (Fig. 5F). T-bet expression was maintained uniformly over the 10 days of culture. RORγt expression was also maintained, although to a lesser degree in some of the cells, most of which corresponded to the IFN-γ-only cells (data not shown).

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Figure 5. Ex vivo isolated murine Th1/Th17 cells coexpress RORγt and T-bet. (A) EAE was induced in C57BL/6 mice and cytokine expression on day 7 after immunization was measured in CD4+ T cells from spleen and lymph nodes after stimulating the cells with PMA/Iono for 4 h. The cells were analyzed before any clinical symptoms of disease were measurable. (B) IFN-γ+IL-17, IL-17+IFN-γ and IL-17+IFN-γ+ CD4+ T cells were isolated combining the IL-17 and IFN-γ secretion assay. (C) RORγt and T-bet were stained intracellularly in the different subsets. (D) mRNA was isolated from ex vivo isolated IL-17+, IL-17+IFN-γ+ and IFN-γ+ Th cells. Relative expression of RORγt and T-bet was determined by real-time PCR. Data show mean±SD of three independent experiments. (E) IL-17+IFN-γ+ CD4+ Th cells were stimulated with anti-CD3/anti-CD28/APC and cultured under neutral conditions (anti-IL-4, anti-IFN-γ and anti-IL-12) for 5 days. The cells were restimulated with PMA/Iono for 4 h and cytokine, T-bet and RORγt expression was measured. (F) The cells from (E) were restimulated with anti-CD3/anti-CD28/APC and cultured under neutral conditions (anti-IL-4, anti-IFN-γ and anti-IL-12) for another 5 days. Cytokine, T-bet and RORγt expression was measured after restimulation with PMA/Iono for 4 h. Data are representative of five independent experiments.

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We have also directly isolated Th1/Th17 cells ex vivo from human blood, and analyzed the stability of RORγt and T-bet expression, as well as IL-17 and IFN-γ re-expression in vitro. Th1/Th17 cells were isolated according to coexpression of IFN-γ and IL-17, using the IFN-γ/IL-17 double secretion assay (Fig. 6A). Viable Th1/Th17 cells were enriched from 1% of CD4+ cells to 94% purity, and these cells expressed both RORγt and T-bet (Fig. 6A and B). Th cells expressing neither cytokine expressed neither RORγt nor T-bet. IFN-γ-only positive cells expressed only T-bet and IL-17-only positive T cells expressed only RORγt (Supporting Information Fig. 6). Directly ex vivo isolated Th1/Th17 cells were then cultured under neutral conditions for 5 or 10 days. Upon restimulation with PMA/ionomycin, about 40% of the cells coexpressed IL-17 and IFN-γ, 26% only IFN-γ and 10% only IL-17 (Fig. 6C). Again, more than 50% of the cells uniformly maintained coexpression of T-bet and RORγt, the canonical Th1 and Th17 master transcription factors over the 10 days of in vitro culture (Fig. 6D).

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Figure 6. Ex vivo isolated human Th1/Th17 cells coexpress RORγt and T-bet. (A) Human CD4+ T cells coexpressing IL-17 and IFN-γ were isolated from peripheral blood using the IL-17 and IFN-γ secretion assays and expression of RORγt and T-bet was measured. Data are representative of five independent experiments. (B) mRNA was isolated from ex vivo isolated IL-17+, IL-17+IFN-γ+ and IFN-γ+ human Th cells. Relative expression of RORγt and T-bet was determined by real-time PCR. Data show mean±SD of four independent experiments. (C) IL-17+IFN-γ+ CD4+ Th cells were cultured under neutral conditions (anti-IL-4 and anti-IFN-γ) for 5 days. The cells were restimulated with PMA/Iono for 6 h and cytokine, T-bet and RORγt expression was measured. (D) The cells from (B) were stimulated with anti-CD3/anti-CD28 and cultured under neutral condition (anti-IL-4 and anti-IFN-γ) for another 5 days. IL-17, IFN-γ, RORγt and T-bet expression was assessed by intracellular staining. Data are representative of five independent experiments. Gating strategy is shown in Supporting Information Fig. 1E.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We here provide a molecular mechanism for the generation of a distinct Th-cell population characterized by the additive phenotypes of Th1 and Th17 cells, the Th1/Th17 cells. Th1/Th17 cells are characterized by the coexpression of the cytokines IFN-γ and IL-17 and the lineage-defining and -determining transcription factors T-bet and RORγt. Th1/Th17 cells develop from Th17 cells upon synergistic action of IFN-γ, required for the upregulation of the IL-12Rβ2 chain and IL-12. IL-12Rβ2 signaling is required for stable imprinting of Th1 cells 20.

The differentiation and maintenance of Th lineage phenotypes, in particular their memory for cytokine expression has been intensely studied 26, 27. Several mechanisms have been identified, such as the inhibition of the Th2 master transcription factor GATA-3 through T-bet-mediated phosphorylation 28, the GATA-3-mediated downregulation of the IL-12Rβ2 chain 29 or STAT4 30, or the sequestering of cytokine genes into heterochromatin 31, which restrict the plasticity of differentiated Th lineage cells. This has led to the overall picture that once fully differentiated cells of the various Th lineages are committed and resistant to conversion into other lineages 32. We have shown previously that Th17 cells which have been generated in vivo are refractory to Th1 and Th2 polarization ex vivo17, whereas in vitro-generated murine Th17 cells and human Th17 cells show a great degree of plasticity 19, 33. To clarify this apparent discrepancy, here we demonstrate that in vivo generated Th17 cells have downregulated the IL-12Rβ2 chain and do not respond to IL-12. However, they still express the IFN-γ receptor and respond to IFN-γ signaling. IFN-γ signaling induces the upregulation of expression of T-bet and of the IL-12Rβ2 chain 20, 24, 34. In this regard, in vivo generated Th17 cells behave like naïve Th cells 20. Naïve Th cells were shown to require sequential activation with IFN-γ and IL-12, resulting in two waves of T-bet induction, whereby IL-12-induced T-bet is required for induction of IFN-γ expression and imprinting of the Ifnγ gene for re-expression 20. Unlike naïve Th cells, in vivo generated Th17 cells maintain their enhanced expression of RORγt and expression of IL-17.

It remains to be shown how expression of the IL-12Rβ2 chain is downregulated in Th17 cells in vivo. Evidence has been provided that IL-17 itself directly 35, or indirectly, by inducing APC to release as yet unidentified factors 36, downregulates IL-12Rβ2 chain expression in activated Th cells. For in vitro-generated and -maintained Th17 cells, we could not confirm these observations. Neither was expression of the IL-12Rβ2 chain downregulated on such cells, nor did blocking of IL-17 upregulate its expression (data not shown).

The downregulation of the IL-12Rβ2 chain also apparently precludes Th1 polarization of Th2 cells. It has been shown for murine and human Th2 cells that IFN-γ and IL-12 in synergy can convert them into Th2/Th1 cells 25, 37, 38, enabling the immune system to combine properties of both Th lineages in individual cells in immune responses to viruses 37 or intracellular bacteria 39. Suryani and Sutton have suggested that IL-17/IFN-γ coexpressing cells are derived from Th1 cells, gaining the ability to express IL-17 40. Although we do not exclude this option here, the conversion of Th1 cells into Th1/Th17 cells must require signals different from the canonical Th17 differentiation signals TGF-β and IL-6. Neither in vitro-generated Th1 cells 17, nor ex vivo isolated Th1 cells, as we show here (Supporting Information Fig. 7), can be induced to express IL-17, by combined action of TGF-β, IL-6 and IL-23. In addition, upon adoptive transfer, Th1 cells are not converted into Th1/Th17 cells in vivo41.

The physiological advantage of Th1/Th17 cells over Th1 and Th17 cells could be their combined effector repertoire on the single-cell level, coexpressing IFN-γ and IL-17, but also chemokine receptors of both Th1 and Th17 cells 41, 42, i.e. CCR2, CCR5 and CXCR3 of Th1 and CCR4 and CCR6 of Th17 cells, allowing them to deliver their cytokines at noncanonical locations. For example, Th1/Th17 cells could deliver IL-17 into inflamed tissue, attracted by the CXCR3 ligands CXCL9, 10 or 11 43. Indeed, it has been suggested in recent publications that Th17 cells expressing IFN-γ have pro-inflammatory properties clearly distinct from Th1 or Th17 cells. Th1/Th17 cells could more efficiently induce the expression of inflammatory mediators on epithelial cells 44 and were found to preferentially accumulate in the CNS of MS–infected patients and EAE mice compared with IFN-γ or IL-17 single producers 45.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Mice and murine antibodies

Specific-pathogen-free 6- to 8-wk old, ex-breeder BALB/c and C57BL/6J mice were obtained from Charles River (Sulzburg, Germany). Mice were sacrificed by cervical dislocation. All animal experiments were performed in accordance with institutional, state and federal guidelines. Anti-IL-4 (11B11), anti-IL-12 (C17.18), anti-IFN-γ (AN17.18.24) antibodies purified from hybridoma supernatants at the Deutsches Rheuma-Forschungszentrum (DRFZ, Berlin, Germany) were used at a final concentration of 20 μg/mL. FITC-conjugated anti-CD4 (GK1.5, Miltenyi Biotec), Alexa Flour 405-conjugated anti-CD4 (GK1.5, purified and coupled at the DRFZ) and PE-conjugated anti-IL-12Rb2 (Clone 305719, R&D Systems, Minneapolis, MN, USA) were used for surface staining. FITC-conjugated anti-IL-17 (eBio17B7; eBioscience) Alexa Flour 647-conjugated anti-T-Bet (eBio4B10, eBioscience), PE-conjugated anti-RORγt (AFKJS-9, eBioscience) were used for intracellular stainings. For STAT4 phosphorylation (pSTAT4) and phosphorylated STAT1 (pSTAT1) staining, we used an Alexa Fluor 647-conjugated pSTAT4 (pY693) and pSTAT1 (pY701) antibody, respectively (both from BD Biosciences).

Murine cell culture conditions and Th differentiation in vitro

Naïve CD4+CD62L+ cells from 6- to 8-wk-old BALB/c mice were isolated as described previously 46. All cultures and assays were performed in RPMI supplemented with 10% fetal bovine serum (Sigma), 100 U/mL penicillium, 0.1 mg/mL streptomycin, 0.3 mg/mL glutamine (Invitrogen) and 10 μM β-mercaptoethanol at 37°C in 5% CO2. Cell cultures were set up at 106 cells/mL and stimulated in the presence of 1 μg/mL anti-CD3 (145-2C11, BD Biosciences), 1 μg/mL anti-CD28 (37.51) and irradiated (30 Gy) splenocytes isolated from BALB/c mice. For Th17 differentiation, cells were stimulated in the presence of TGF-β1 (1 ng/mL), IL-6 (20 ng/mL), IL-23 (20 ng/mL) (all from R&D Systems), anti-IL-4 and anti-IFN-γ. Where indicated, recombinant IL-12 (5 ng/mL; R&D Systems) and recombinant IFN-γ (10 ng/mL) were added. The IL-17-producing cells were isolated as described previously 17.

Isolation of cells according to IL-12Rβ2 expression

IL-12Rβ2 was stained according to the manufactor's instructions. IL-12Rβ2 high and low cells were sorted on a FACS Aria (BD Biosciences). Sorted cells were stimulated with 0.1 ng/mL PMA and 1 μg/mL ionomycin (Sigma-Aldrich) for 4 h. Briefly, 5 μg/mL Brefeldin A (Sigma Chemicals) was added after 1 h. The cells were stained for intracellular cytokines, as described previously 47. Absolute cell numbers and cytokine expression were determined with a MACSQuant Analyzer (Miltenyi Biotec).

Isolation of IL-17- and IL-17/IFN-γ-secreting cells ex vivo

IL-17-producing cells were isolated ex vivo as described previously 17. CD4+ T cells were isolated with anti-CD4 magnetic beads (Miltenyi Biotec) and stimulated with 0.1 ng/mL PMA and 1 μg/mL ionomycin for 2 h. IL-17+ IFN-γ+ T cells were isolated by combining the IL-17 PE and IFN-γ APC Cytokine Secretion Assay & Detection Kits (Miltenyi Biotech) and sorting with a FACSAria (BD Biosciences).

Isolation of human IL-17-secreting cells ex vivo

PBMC were obtained from buffy coats of healthy donors (German Red Cross) by Ficoll-Hypaque gradient (Sigma-Aldrich). All experiments with human material were approved by the local ethics committee. CD4+ T cells were isolated with anti-CD4 magnetic beads (Miltenyi Biotec) and stimulated with 0.1 ng/mL PMA and 1 μg/mL ionomycin for 4 h. IL-17+ IFN-γ+ CD4+ T cells were stained by combining the human IL-17 and IFN-γ secretion assays (Miltenyi Biotech) and sorted with a FACSAria (BD Biosciences). For intracellular cytokine staining, anti-CD4-Alexa Fluor 405 (TT1), anti-IL-17A-Alexa Fluor 647 (eBioscience) and anti-IFN-γ-FITC (4SB3) were used. Isolated cells were cultured in vitro with 0.1 μg/mL anti-CD3 and 6 μg/mL anti-CD28 antibodies (BD Biosciences). Cytokines were used at either 10 ng/mL (IL-12 and IFN-γ, BD Biosciences), or 1000 U/mL (IL-2, Chiron). Neutralizing antibodies to IFN-γ (Peprotech) were used at 10 μg/mL.

EAE induction

EAE was induced in 8- to 12-wk-old C57BL/6 mice by subcutaneous injection of 200 μg myelin oligodendrocyte glycoprotein35–55 peptide 48 (mevgwyrspfsrvvhlyrngk; synthesized by Dr. R. Volkmer, Charité, Berlin, Germany) emulsified in complete Freund's adjuvant (containing 1 mg/mL Mycobacterium tuberculosis H37RA, Sigma) together with i.v. administration of 200 ng pertussis toxin (Sigma) on days 0 and 2. Disease severity was assigned scores daily on a scale of 0–5 as follows: 0, no paralysis; 1, limp tail; 2, limp tail and partial hindleg paralysis; 3, complete hindleg paralysis; 4, tetraparesis and 5, moribund.

Flow cytometry

In brief, 5×106 cells/mL were stimulated in RPMI medium with PMA and Ionomycin (Sigma) for 4 h. In total, 5 μg/mL Brefeldin A was added after 1 h.

For intracellular T-bet and RORγt staining, we used the Foxp3 staining buffer set (eBioscience) according to the manufacturer's instructions. Mouse IgG1, κ (eBioscience) was used as Isotype Control for T-bet. CD4+CD62L+ were stained as negative control for RORγt. Intracellular IL-17 and IFN-γ in murine and human cells were stained under the same conditions. For pSTAT4 and pSTAT1 staining, we used Phosflow Lyse/Perm buffer and Perm Buffer III (BD Biosciences) according to the manufacturer's instruction. The cells were stimulated with IL-12 (10 ng/mL) for 30 min for pSTAT4 and with IFN-γ (10 ng/mL) for 15 min for pSTAT1 staining. Flow cytometric analysis was performed with LSRII and FACSCalibur, using CellQuest and FlowJo (Tree Star, Ashland, OR, USA) software.

RNA quantification

RNA preparation and cDNA synthesis was performed as described previously 46. For normalization of murine and human cDNA, the transcripts for the housekeeping genes hypoxanthine guanine phosphoribosyl transferase and ubiquitin ligase H5 (UbcH5) were quantified, respectively. Murine primer sets for the real-time PCR were as previously described 17 except for: murine IL-12Rβ2 F: 5′-CTgATCCTCCATTACAgAA-3′, IL-12Rβ2 R: 5′-CggAAgTAACgAATTgAgAA-3′, murine IFN-γ R2F: 5′-CCgAgTgAAgTACTggTTTC-3′ and IFN-γ R2R: 5′-gTgTTTggAgCACATCATC-3′. Human Primer Sets were as follows:

UbcH5 F: 5′-TCTTGACAATTCATTTCCCAACAG-3′, UbcH5 R 5′-TCAGGCACTAAAGGA TCATCTGG-3′, RORγt F: 5′-gAggAAgTCCATgTgggAgA-3′, RORγt R: 5′- TCCTAACCAgCACCACTTCC-3′, T-bet F: 5′-CCCCGGCTGCATATCG-3′ and T-bet R: 5′-ATCCTTTGGCAAAGGGGTTA-3′.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank Jenny Kirsch and Toralf Kaiser for excellent cell sorting. This work has been supported by the German Science Foundation (DFG SFB421, SFB618 and SFB TR52) and MUGEN-Network of Excellence. I. L. was funded by the Berlin-Brandenburg School of Regenerative Therapies (BSRT).

Conflict of interest: Anne Richter is a paid employee of Miltenyi Biotec. The other authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
  • 1
    Khoruts, A., Miller, S. D. and Jenkins, M. K., Neuroantigen-specific Th2 cells are inefficient suppressors of experimental autoimmune encephalomyelitis induced by effector Th1 cells. J. Immunol. 1995. 155: 50115017.
  • 2
    Yang, Y., Weiner, J., Liu, Y., Smith, A. J., Huss, D. J., Winger, R., Peng, H. et al., T-bet is essential for encephalitogenicity of both Th1 and Th17 cells. J. Exp. Med. 2009. 206: 15491564.
  • 3
    Luger, D., Silver, P. B., Tang, J., Cua, D., Chen, Z., Iwakura, Y., Bowman, E. P. et al., Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J. Exp. Med. 2008. 205: 799810.
  • 4
    Tarrant, T. K., Silver, P. B., Chan, C.-C., Wiggert, B. and Caspi, R. R., Endogenous IL-12 is required for induction and expression of experimental autoimmune uveitis. J. Immunol. 1998. 161: 122127.
  • 5
    Powrie, F., Leach, M. W., Mauze, S., Menon, S., Caddle, L. B. and Coffman, R. L., Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+T cells. Immunity 1994. 1: 553562.
  • 6
    Cua, D. J., Sherlock, J., Chen, Y., Murphy, C. A., Joyce, B., Seymour, B., Lucian, L. et al., Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003. 421: 744748.
  • 7
    Yen, D., Cheung, J., Scheerens, H., Poulet, F., McClanahan, T., McKenzie, B., Kleinschek, M. A. et al., IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 2006. 116: 13101316.
  • 8
    Kolls, J. K. and Linden, A., Interleukin-17 family members and inflammation. Immunity 2004. 21: 467476.
  • 9
    Stout, R. D. and Bottomly, K., Antigen-specific activation of effector macrophages by IFN-gamma producing (TH1) T cell clones. Failure of IL-4-producing (TH2) T cell clones to activate effector function in macrophages. J. Immunol. (Baltimore, MD) 1989. 142: 760765.
  • 10
    O'Connor, R. A., Prendergast, C. T., Sabatos, C. A., Lau, C. W., Leech, M. D., Wraith, D. C. and Anderton, S. M., Cutting edge: Th1 cells facilitate the entry of Th17 cells to the central nervous system during experimental autoimmune encephalomyelitis. J. Immunol. 2008. 181: 37503754.
  • 11
    Lees, J. R., Iwakura, Y. and Russell, J. H., Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines. J. Immunol. (Baltimore, MDd) 2008. 180: 80668072.
  • 12
    Infante-Duarte, C., Horton, H. F., Byrne, M. C. and Kamradt, T., Microbial lipopeptides induce the production of IL-17 in Th cells. J. Immunol. 2000. 165: 61076115.
  • 13
    Aarvak,T., Chabaud, M., Miossec, P. and Natvig, J. B., IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells. J. Immunol. 1999. 162: 12461251.
  • 14
    Annunziato, F., Cosmi, L., Santarlasci, V., Maggi, L., Liotta, F., Mazzinghi, B., Parente, E. et al., Phenotypic and functional features of human Th17 cells. J. Exp. Med. 2007. 204: 18491861.
  • 15
    Gorelik, L., Constant, S. and Flavell, R. A., Mechanism of transforming growth factor beta-induced inhibition of T helper type 1 differentiation. J. Exp. Med. 2002. 195: 14991505.
  • 16
    Harrington, L. E., Hatton, R. D., Mangan, P. R., Turner, H., Murphy, T. L., Murphy, K. M. and Weaver, C. T., Interleukin 17-producing CD4+effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. 2005. 6: 11231132.
  • 17
    Lexberg, M. H., Taubner, A., Forster, A., Albrecht, I., Richter, A., Kamradt, T., Radbruch, A. and Chang, H. D., Th memory for interleukin-17 expression is stable in vivo. Eur. J. Immunol. 2008. 38: 26542664.
  • 18
    Cosmi, L., Maggi, L., Santarlasci, V., Capone, M., Cardilicchia, E., Frosali, F., Querci, V. et al., Identification of a novel subset of human circulating memory CD4(+) T cells that produce both IL-17A and IL-4. J. Allergy Clin. Immunol. 2010. 125: 222230; e221–e224.
  • 19
    Lee, Y. K., Turner, H., Maynard, C. L., Oliver, J. R., Chen, D., Elson, C. O. and Weaver, C. T., Late developmental plasticity in the T helper 17 lineage. Immunity 2009. 30: 92107.
  • 20
    Schulz, E. G., Mariani, L., Radbruch, A. and Hofer, T., Sequential polarization and imprinting of type 1 T helper lymphocytes by interferon-gamma and interleukin-12. Immunity 2009. 30: 673683.
  • 21
    van Rietschoten, J. G., Smits, H. H., van de Wetering, D., Westland, R., Verweij, C. L., den Hartog, M. T. and Wierenga, E. A., Silencer activity of NFATc2 in the interleukin-12 receptor beta 2 proximal promoter in human T helper cells. J. Biol. Chem. 2001. 276: 3450934516.
  • 22
    Sanceau, J., Merlin, G. and Wietzerbin, J., Tumor necrosis factor-alpha and IL-6 up-regulate IFN-gamma receptor gene expression in human monocytic THP-1 cells by transcriptional and post-transcriptional mechanisms. J. Immunol. 1992. 149: 16711675.
  • 23
    Pernis, A., Gupta, S., Gollob, K. J., Garfein, E., Coffman, R. L., Schindler, C. and Rothman, P., Lack of interferon gamma receptor beta chain and the prevention of interferon gamma signaling in TH1 cells. Science 1995. 269: 245247.
  • 24
    Afkarian, M., Sedy, J. R., Yang, J., Jacobson, N. G., Cereb, N., Yang, S. Y., Murphy, T. L. and Murphy, K. M., T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+T cells. Nat. Immunol. 2002. 3: 549557.
  • 25
    Szabo, S. J., Dighe, A. S., Gubler, U. and Murphy, K. M., Regulation of the interleukin (IL)-12R beta 2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 1997. 185: 817824.
  • 26
    Lohning, M., Richter, A. and Radbruch, A., Cytokine memory of T helper lymphocytes. Adv. Immunol. 2002. 80: 115181.
  • 27
    Ansel, K. M., Lee, D. U. and Rao, A., An epigenetic view of helper T cell differentiation. Nat. Immunol. 2003. 4: 616623.
  • 28
    Hwang, E. S., Szabo, S. J., Schwartzberg, P. L. and Glimcher, L. H., T helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science 2005. 307: 430433.
  • 29
    Ouyang, W., Ranganath, S. H., Weindel, K., Bhattacharya, D., Murphy, T. L., Sha, W. C. and Murphy, K. M., Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 1998. 9: 745755.
  • 30
    Usui, T., Nishikomori, R., Kitani, A. and Strober, W., GATA-3 suppresses Th1 development by downregulation of Stat4 and not through effects on IL-12Rbeta2 chain or T-bet. Immunity 2003. 18: 415428.
  • 31
    Grogan, J. L., Wang, Z. E., Stanley, S., Harmon, B., Loots, G. G., Rubin, E. M. and Locksley, R. M., Basal chromatin modification at the IL-4 gene in helper T cells. J. Immunol. 2003. 171: 66726679.
  • 32
    Murphy, E., Shibuya, K., Hosken, N., Openshaw, P., Maino, V., Davis, K., Murphy, K. and O'Garra, A., Reversibility of T helper 1 and 2 populations is lost after long-term stimulation. J. Exp. Med. 1996. 183: 901913.
  • 33
    Annunziato, F., Cosmi, L. and Romagnani, S., Human and murine Th17. Curr. Opin. HIV AIDS 2010. 5: 114119.
  • 34
    Mullen, A. C., High, F. A., Hutchins, A. S., Lee, H. W., Villarino, A. V., Livingston, D. M., Kung, A. L. et al., Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science 2001. 292: 19071910.
  • 35
    Toh, M.-L., Kawashima, M., Zrioual, S., Hot, A., Miossec, P. and Miossec, P., IL-17 inhibits human Th1 differentiation through IL-12R[beta]2 downregulation. Cytokine 2009. 48: 226.
  • 36
    Nakae, S., Iwakura, Y., Suto, H. and Galli, S. J., Phenotypic differences between Th1 and Th17 cells and negative regulation of Th1 cell differentiation by IL-17. J. Leukoc. Biol. 2007. 81: 12581268.
  • 37
    Hegazy, A. N., Peine, M., Helmstetter, C., Panse, I., Frohlich, A., Bergthaler, A., Flatz, L. et al., Interferons direct Th2 cell reprogramming to generate a stable GATA-3(+)T-bet(+) cell subset with combined Th2 and Th1 cell functions. Immunity 2010. 32: 116128.
  • 38
    Fili, L., Ferri, S., Guarna, F., Sampognaro, S., Manuelli, C., Liotta, F., Cosmi, L. et al., Redirection of allergen-specific TH2 responses by a modified adenine through Toll-like receptor 7 interaction and IL-12/IFN release. J. Allergy Clin. Immunol. 2006. 118: 511517.
  • 39
    Lin, Y., Ritchea, S., Logar, A., Slight, S., Messmer, M., Rangel-Moreno, J., Guglani, L. et al., Interleukin-17 is required for T helper 1 cell immunity and host resistance to the intracellular pathogen Francisella tularensis. Immunity 2009. 31: 799810.
  • 40
    Suryani, S. and Sutton, I., An interferon-gamma-producing Th1 subset is the major source of IL-17 in experimental autoimmune encephalitis. J. Neuroimmunol. 2007. 183: 96103.
  • 41
    Lim, H. W., Lee, J., Hillsamer, P. and Kim, C. H., Human Th17 cells share major trafficking receptors with both polarized effector T cells and FOXP3+regulatory T cells. J. Immunol. 2008. 180: 122129.
  • 42
    Annunziato, F., Cosmi, L., Liotta, F., Maggi, E. and Romagnani, S., The phenotype of human Th17 cells and their precursors, the cytokines that mediate their differentiation and the role of Th17 cells in inflammation. Int. Immunol. 2008. 20: 13611368.
  • 43
    Janke, M., Peine, M., Nass, A., Morawietz, L., Hamann, A. and Scheffold, A., In vitro-induced Th17 cells fail to induce inflammation in vivo and show an impaired migration into inflamed sites. Eur. J. Immunol. 2010. 40: 10891098.
  • 44
    Boniface, K., Blumenschein, W. M., Brovont-Porth, K., McGeachy, M. J., Basham, B., Desai, B., Pierce, R. et al., Human Th17 cells comprise heterogeneous subsets including IFN-gamma-producing cells with distinct properties from the Th1 lineage. J. Immunol. 2010. 185: 679687.
  • 45
    Kebir, H., Ifergan, I., Alvarez, J. I., Bernard, M., Poirier, J., Arbour, N., Duquette, P. and Prat, A., Preferential recruitment of interferon-gamma-expressing T H 17 cells in multiple sclerosis. Ann. Neurol. 2009. 66: 390402.
  • 46
    Chang, H. D., Helbig, C., Tykocinski, L., Kreher, S., Koeck, J., Niesner, U. and Radbruch, A., Expression of IL-10 in Th memory lymphocytes is conditional on IL-12 or IL-4, unless the IL-10 gene is imprinted by GATA-3. Eur. J. Immunol. 2007. 37: 807817.
  • 47
    Murphy, K. M., Heimberger, A. B. and Loh, D. Y., Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 1990. 250: 17201723.
  • 48
    Brustle, A., Heink, S., Huber, M., Rosenplanter, C., Stadelmann, C., Yu, P., Arpaia, E. et al., The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4. Nat. Immunol. 2007. 8: 958966.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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