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Abstract
Objective
IL-17-producing T cells (Th17 cells) have been identified as a distinct lineage of CD4+ T helper cells in mice. Since this discovery, many efforts have been made to investigate the characteristics and the role of human Th17 cells and the factors involved in their differentiation. This study was undertaken to assess the effects of cytokines and stimulatory conditions on the differentiation of human CD4+ T cells into Th17 cells.
Methods
Peripheral blood CD4+ T cells were isolated from healthy humans and then these cells were cultured with using various stimulatory conditions. The Th17 cells and regulatory T (Treg) cells were detected by flow cytometry (FACs). The related gene expressions of cytokines, transcription factors and chemokine receptors were determined by ELISA and RT-PCR.
Results
In the presence of inflammatory cytokines, TNFa and IL-1b, the human CD4+ T cells rapidly produced IL-17 in response to anti-CD3/anti-CD28 stimulation, whereas, with anti-CD3/ anti-CD28 stimulation alone, the CD4+ T cells expressed low levels of IL-17. TNFa and IL-1b were also important inducers of IL-22 production. IL-6 and IL-23 up-regulated the RORgammat, CCR4 and CCR6 expressions in the human CD4+ T cells. In response to TGF-b and IL-2, the human CD4+ T cells were rapidly induced to express FoxP3, IL-10 and CCR7, as compared with anti-CD3/anti-CD28 stimulation alone.
Conclusion
The effect of inflammatory cytokines on the differentiation of human Th17 cells may help us to understand their pathogenic role. Moreover, the differential expression of chemokine receptors and transcription factors of the subsets of CD4+ T cells with the different features of Th17 and Treg, may raise new issues concerning the pathogenesis of autoimmune inflammatory diseases.
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Fig. 1.
Th17 cell differentiation in human CD4＋ T cells. (A) Flow cytometry of intracellular human IL-17 producing CD4＋ T cells from peripheral blood. CD4＋ T cells cultured with plate bound anti-CD3, anti-CD28 and various cytokines in vitro for 6d. Neutralize antibodies used anti-IFN-γ and anti-IL-4. Data are representative of ten independent experiments. (B) ELISA quantification of IL-22 and IL-17 in culture supernatants of A. Data are representative of ten independent experiments. Values are mean±SD. ∗p＜0.01.
Fig. 2.
Expression of transcription factors in human CD4＋ Th17 cells. RT-PCR analysis of T-bet, GATA3 and RORc expression in CD4＋ T cells. CD4＋ T cells cultured with plate bound anti-CD3, anti-CD28 and various cytokines in vitro for 3d. β −actin was used as a loading control. Results were normalized for β-actin expression. Statistical data repeat of three independent experiments. Values are mean±SD. ∗p＜0.01.
Fig. 3.
Expression of chemokines receptors in human CD4＋ Th17 cell. RT-PCR analysis of CXCR3, CCR7, CCR4, and CCR6 expression in CD4＋ T cells. CD4＋ T cells cultured with plate bound anti-CD3, anti-CD28 and various cytokines in vitro for 3d. Results were normalized for β-actin expression. Statistical data repeat of three independent experiments. Values are mean±SD. ∗p＜0.01.
Fig. 4.
Th17 versus Treg lineages are differential expression of cytokine and chemokines receptors in human CD4＋ T cells. (A) Flow cytometry of intracellular human Foxp3 expression CD4＋ CD25＋ T cells from peripheral blood. CD4＋ T cells cultured with plate bound anti-CD3, anti-CD28 and various cytokines in vitro for 3d (Th0: anti-CD3 and anti-CD28, Th17: anti-IFN-γ, anti-IL-4, TGF-β, IL-6, IL-23, IL-1β and TNF-α, Treg: anti-IFN-γ, anti-IL-4, TGF-β and IL-2). Data are representative of ten independent experiments. (B) ELISA quantification of IL-22, IL-17 and IL-10 in culture supernatants of A. Data are representative of ten independent experiments. Values are mean±SD. (C) RT-PCR analysis of CXCR3, CCR4, CCR6, and CCR7 expression in CD4＋ T cells. CD4＋ T cells cultured with plate bound anti-CD3, anti-CD28 and various cytokines in vitro for 3d. β-actin was used as a loading control. Results were normalized for β-actin expression. Statistical data repeat of three independent experiments. Values are mean±SD. ∗p＜0.01 versus Th0 or Th17.
Table 1.
Primers used in RT-PCR
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