Development and characterization of IL-21-producing CD4+ T cells - PubMed (original) (raw)

Development and characterization of IL-21-producing CD4+ T cells

Akira Suto et al. J Exp Med. 2008.

Abstract

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells, functions as an autocrine growth factor for Th17 cells, and plays critical roles in autoimmune diseases. In this study, we investigated the differentiation and characteristics of IL-21-producing CD4(+) T cells by intracellular staining. Unexpectedly, we found that under Th17-polarizing conditions, the majority of IL-21-producing CD4(+) T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21-producing CD4(+) T cells without the induction of IL-4, IFN-gamma, IL-17A, or IL-17F production. On the other hand, TGF-beta inhibited IL-6- and IL-21-induced development of IL-21-producing CD4(+) T cells. IL-2 enhanced the development of IL-21-producing CD4(+) T cells under Th17-polarizing conditions. Finally, IL-21-producing CD4(+) T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6, but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21-producing CD4(+) T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6-rich environment devoid of TGF-beta, and that IL-21 functions as an autocrine growth factor for IL-21-producing CD4(+) T cells.

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Figures

Figure 1.

Figure 1.

Establishment of a single-cell analysis of IL-21–producing cells. (A) IL-21 and -17A are produced by activated CD4+ T cells under Th17-polarizing conditions. Purified CD4+ T cells from lymph nodes of C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-, Th2-, and Th17-polarizing conditions. On day 5, 106 cells were restimulated with anti-CD3 mAb/anti-CD28 mAb for 24 h. The levels of cytokines in the culture supernatants were measured by ELISA. Data are the means ± the SD from four independent experiments. *, P < 0.01. (B) Establishment of a single-cell analysis of IL-21–producing cells. Ba/F3 cells were infected with pMX-IL-21-IRES-GFP retrovirus or control retrovirus (pMX-IRES-GFP). Nine clones of pMX-IL-21-IRES-GFP retrovirus-infected Ba/F3 cells (Ba/F3-IL-21-GFP cells) and one clone of control retrovirus-infected Ba/F3 cells (Ba/F3-GFP cells) were selected by limiting dilution. IL-21 in the culture supernatants was measured by ELISA (left). Ba/F3-GFP cells and Ba/F3-IL-21-GFP clone #6 cells were fixed, permeabilized, and incubated with IL-21R-Fc or PBS. After washing, cells were visualized with anti-Fc PE (right).

Figure 2.

Figure 2.

CD4+ T cells producing IL-21, but not IL-17A/IL-17F, are present under Th17-polarizing conditions. Naive CD4+ T cells from lymph nodes of C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-, Th2-, and Th17-polarizing conditions for 5 d. Cells were evaluated for the expression of the indicated cytokines by intracellular cytokine staining as described in the Materials and methods. Data are representative of three independent experiments.

Figure 3.

Figure 3.

IL-6 induces, but TGF-β inhibits, the development of IL-21–producing CD4+ T cells. (A and B) Naive CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of 10 μg/ml anti–IL-4 mAb and 10 μg/ml anti–IFN-γ mAb (neutral condition) with or without 100 ng/ml IL-6. Where indicated, 0.2–2 ng/ml TGF-β was added. (A) 4 d later, cells were stimulated with PMA/ionomycin, and intracellular staining for the indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments. (B) 4 d later, cells were washed and stimulated with PMA/ionomycin for 8 h at 2 × 106 cells/ml. The levels of IL-21 in the culture supernatants were measured by ELISA. Data are the mean ± the SD (n = 3). *, P < 0.05; **, P < 0.01.

Figure 4.

Figure 4.

Kinetics of IL-21 production from CD4+ T cells. Naive CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb (neutral condition) with 100 ng/ml IL-6 or IL-6 plus 1 ng/ml TGF-β. At indicated times after stimulation, cells were stimulated with PMA/ionomycin, and intracellular staining for the indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments.

Figure 5.

Figure 5.

Smad3 is involved in TGF-β–mediated suppression of IL-21 production from CD4+ T cells. Purified CD4+ T cells from Smad3−/− mice or littermate WT mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb, anti–IFN-γ mAb, and anti–IL-2 mAb with 100 ng/ml IL-6 or IL-6 plus 1 or 5 ng/ml TGF-β for 3 d. Cells were then stimulated with PMA/ionomycin and intracellular staining for IL-21 versus IL-17A was performed. Shown are representative FACS profiles from three independent experiments.

Figure 6.

Figure 6.

IL-2 does not inhibit the development of IL-21–producing CD4+ T cells. Naive CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb with 100 ng/ml IL-6 or IL-6 plus 1 ng/ml TGF-β. Where indicated, either 10 ng/ml IL-2 or 10 μg/ml anti–IL-2 antibody was added to the culture. 4 d later, cells were stimulated with PMA/ionomycin and intracellular staining for the indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments.

Figure 7.

Figure 7.

IL-21 induces the development of IL-21–producing CD4+ T cells. (A) Naive CD4+ T cells from IL-21R−/− mice or littermate WT mice on a BALB/c background were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb (neutral condition) with 100 ng/ml IL-21 or 1 ng/ml IL-21 plus TGF-β. 4 d later, cells were stimulated with PMA/ionomycin and intracellular staining for IL-21 versus IL-17A was performed. Shown are representative FACS profiles from three independent experiments. (B) Naive CD4+ T cells from BALB/c mice were stimulated with anti-CD3 mAb/anti-CD28 mAb for 4 d in the presence of anti–IL-4 mAb and anti–IFN-γ mAb with IL-6 or IL-6 plus TGF-β. Where indicated, soluble IL-21R-Fc (10 μg/ml) was added. Cells were stimulated with PMA/ionomycin and intracellular staining for IL-21 versus IL-17A was performed. Shown are representative FACS profiles from three independent experiments. (C) Naive CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th2-polarizing conditions for 5 d. Where indicated, a neutralizing antibody against 10 μg/ml IL-6 and/or soluble IL-21R-Fc were added. Cells were then stimulated with PMA/ionomycin and intracellular staining for the indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments.

Figure 8.

Figure 8.

Expression of transcription factors in CD4+ T cells cultured in the presence of IL-6. Naive CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb with IL-6, IL-6 plus TGF-β, or TGF-β alone for 60 h. As controls, naive CD4+ T cells were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-polarizing conditions or Th2-polarizing conditions for 60 h. The expression of RORγt, Foxp3, T-bet, and GATA3 was assessed by real-time PCR. Data are representative of three independent experiments.

Figure 9.

Figure 9.

The frequency of IL-21–producing cells is increased with the rounds of cell cycle progression. Naive CD4+ T cells from C57BL/6 mice were labeled with CFSE, stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb (neutral condition) with IL-6 or IL-6 plus TGF-β for 4 d. Cells were stimulated with PMA/ionomycin and intracellular staining for IL-21, -17A, and -17F was performed. Shown are representative FACS profiles from three independent experiments.

Figure 10.

Figure 10.

IL-21–producing CD4+ T cells do not lose their ability to produce IL-21 when they are restimulated under Th1- or Th2-polarizing conditions. (A) Purified CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb with IL-6 for 5 d. Cells were restimulated for another 5 d under the same condition (top) or Th1-, Th2-, or Th17-polarizing conditions (bottom). On day 10, cells were stimulated with anti-CD3 mAb and intracellular staining for indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments. (B) As positive controls, CD4+ T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-, Th2-, or Th17-polarizing conditions for 5 d. Cells were restimulated under the same conditions for another 5 d. On day 10, cells were stimulated with anti-CD3 mAb and intracellular staining for indicated cytokines was performed.

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