Optimal induction of T helper 17 cells in humans requires T cell receptor ligation in the context of Toll-like receptor-activated monocytes - PubMed (original) (raw)

Optimal induction of T helper 17 cells in humans requires T cell receptor ligation in the context of Toll-like receptor-activated monocytes

Hayley G Evans et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Recently, a new lineage of CD4+ T cells has been described in the mouse that specifically secretes IL-17 [T helper (Th) 17]. This discovery has led to a revision of the hypothesis that many autoimmune diseases are predominantly a Th1 phenomenon and may instead be critically dependent on the presence of Th17 cells. Murine Th17 cells differentiate from naïve T cell precursors in the presence of TGF-beta and IL-6 or IL-21. However, given their putative importance in human autoimmunity, very little is known about the pathways that control the expression of IL-17 in humans. Here we show that the factors that determine the expression of IL-17 in human CD4+ T cells are completely different from mice. IL-6 and IL-21 were unable to induce IL-17 expression in either naïve or effector T cells, and TGF-beta actually inhibited IL-17 expression. The expression of IL-17 was maximally induced from precommitted precursors present in human peripheral blood by cell-cell contact with Toll-like receptor-activated monocytes in the context of T cell receptor ligation. Furthermore, unlike IFN-gamma, IL-17 expression was not suppressed by the presence of FOXP3+ regulatory CD4+ T cells. Taken together, these data indicate that human and mouse Th17 cells have important biological differences that may be of critical importance in the development of therapeutic interventions in diseases characterized by aberrant T cell polarization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Th17 induction in human CD4+ T cells is promoted in the presence of monocytes but inhibited by the addition of IL-6 and TGF-β. (A) Bulk CD4+ T cells (1 × 106 per well) were cultured with anti-CD3/CD28 microbeads or with a 1:1 ratio of irradiated (3,000 Gy), live resting, or live LPS-activated monocytes, in the absence or presence of IL-6 (20 ng/ml) and TGF-β (1 ng/ml). After 6 days the cells were stimulated with PMA/ionomycin followed by Golgistop. Cells were stained with either the appropriate isotype controls or FITC-labeled IFN-γ, PE-labeled IL-17, and PC5-labeled CD14. The percentage of IFN-γ+, IL-17+, or double positive CD4+ T cells is indicated in the quadrants. Monocytes were gated out based on their size and CD14 expression. (B) CD45RA+ naïve (n = 3) and CD45RO+ memory (n = 4) CD4+ T cells were either cocultured at a 1:1 ratio with monocytes in the presence of LPS and anti-CD3 mAb (M + LPS) or cultured alone with IL-6, TGF-β, and anti-CD3 (IL-6/TGF-β). At day 3, cells were stained as described above and the average percentage of IL-17+ T cells (± SEM) is shown. Isotype control staining consistently resulted in <0.1% positive cells throughout the experiments. (C) Bulk CD4+ T cells stimulated and stained directly from blood for IFN-γ and IL-17 (n = 5).

Fig. 2.

Fig. 2.

Optimal Th17 induction in human CD4+ T cells requires cell contact with activated monocytes. Naïve or memory T cells were cultured for 3 days in the presence of anti-CD3 under various conditions and then stained as described in Fig. 1. (A) T cells were cultured in the absence or presence of monocytes, LPS, or both. The average percentage (± SEM) of IL-17+ T cells of two independent experiments is shown. (B) T cells were cultured with LPS either in a coculture with monocytes or in a transwell culture with a membrane separating the T cells and monocytes. The average percentage of IL-17+ cells (± SEM) of four independent experiments is shown. (C) Memory T cells were cultured for 3 days with monocytes and LPS in the presence or absence of LPS, zymosan, flagellin, or LTA. The average ± SEM is shown for flow cytometry (n = 6; *, P < 0.05) and ELISA (n = 4).

Fig. 3.

Fig. 3.

Th1 vs. Th2 skewing conditions have opposite effects on the induction of human Th17 cells. Memory CD4+ T cells (0.5 × 106 cells per well) were cultured with anti-CD3, monocytes, and LPS in the absence or presence of either Th1 (hrIL-12 plus anti-IL-4 mAb) or Th2 (hrIL-4 plus anti-IFN-γ mAb) skewing conditions for 3 days. Cells were then stimulated and stained as described in Fig. 1. (A) A representative flow cytometry plot. (B) The average percentage (± SEM) of IFN-γ or IL-17 single positive cells (n = 7). (C) Analysis of cell culture supernatants by ELISA (n = 6). (D) Cells were cultured as above, RNA was extracted by using TRIzol reagent, and cDNA was produced by iScript kit reaction. Shown is relative abundance (2ΔΔCT) of IL-17 in all three conditions. Reactions were normalized to β-actin, and monocytes alone were used as a baseline for calculation. (E) Memory CD4+ T cells were cultured either alone or in the presence or absence of IL-4, anti-IFN-γ mAb, or both. Cells were then stimulated and analyzed by flow cytometry. A representative from four experiments is shown.

Fig. 4.

Fig. 4.

CD4+CD25+Foxp3+ Tregs promote the induction of IL-17+ T cells by suppressing Th1 responses. Memory CD4+ T cells (0.5 × 106 cells per well) were cultured with anti-CD3, monocytes, and LPS in the absence or presence of CFSE- or DDAO-labeled CD4+CD25+Foxp3+ Tregs at a 1:1 ratio for 3 days, then stimulated and stained as described in Fig. 1. (A) ELISA results for IFN-γ from cultures in the presence or absence of Tregs (**, P < 0.01; n = 8). (B) ELISA results for IL-17 from cultures in the presence or absence of Tregs (n = 7). (C and D) Intracellular IL-17 in the memory T cell pool in the presence and absence of Tregs (*, P < 0.05; n = 8) and as shown by a dot plot of one experiment; Tregs were gated out based on their CFSE or DDAO labeling. Control addition of CFSE/DDAO-labeled CD4+CD45RO+CD25−Foxp3− T cells did not lead to a reduction in IFN-γ or IL-17 (data not shown).

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