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. 2008 Dec 16;105(50):19845-50.
doi: 10.1073/pnas.0806472105. Epub 2008 Dec 4.

"V体育官网入口" Critical role of ROR-γt in a new thymic pathway leading to IL-17-producing invariant NKT cell differentiation

Marie-Laure Michel  1 , Daniella Mendes-da-Cruz, Alexandre Castro Keller, Matthias Lochner, Elke Schneider, Michel Dy, Gérard Eberl, Maria C Leite-de-Moraes
Affiliations

V体育ios版 - Critical role of ROR-γt in a new thymic pathway leading to IL-17-producing invariant NKT cell differentiation

Marie-Laure Michel et al. Proc Natl Acad Sci U S A. .

Abstract

Invariant natural killer T (iNKT) cells constitute a subpopulation of T cells that recognize glycolipids presented by CD1d molecules. They are characterized by their prompt production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma), which enables them to modulate diverse immune responses. Recently, we enlarged this concept by identifying a distinct IL-17-producing iNKT cell subset, named iNKT17 cells. The mechanisms leading to the acquisition of this new iNKT cell activity are unknown. Herein we show that IL-17-producing iNKT cells are already present in the thymus, predominantly among a subset regarded so far as an immature stage of thymic iNKT cell development, the CD1d tetramer(pos)CD44(pos)NK1. 1(neg)CD4(neg) cells. Using EGFP reporter mice, we demonstrate that the transcription factor ROR-gammat is critical for the thymic differentiation of this subset because only ROR-gammat(pos) iNKT cells are capable of massively secreting IL-17. Moreover, IL-17-producing CD1d tetramer(pos)CD44(pos)NK1. 1(neg)CD4(neg) thymic iNKT cells have reached a mature differentiation stage because they fail to generate other cell subsets in fetal thymic organ culture. Conversely, thymic ROR-gammat(neg) iNKT cell precursors give rise to progeny, but acquire neither ROR-gammat expression nor the ability to secrete IL-17. In conclusion, our findings demonstrate an alternative thymic pathway leading to the development of iNKT17 cells that requires ROR-gammat expression. VSports手机版.

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"VSports手机版" Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Thymic iNKT cells produce IL-17. (A) iNKT cell-enriched (CD8neg) thymocytes recovered from C57BL/6 (WT) or Jα18−/− mice were stimulated for 72 h at a concentration of 106 cells /ml with α-GalCer in the presence of irradiated macrophages from Jα18−/− mice. IL-17, IL-4 and IFN-γ were measured in the supernatants. (B and C) CD1d tetramerposCD8neg thymocytes were sorted into 4 distinct subsets or stages (St), namely CD44negNK1.1neg (St 1), CD44posNK1.1negCD4pos (St 2, CD4pos), CD44posNK1.1negCD4neg (St 2, CD4neg), and CD44posNK1.1pos (St 3) as illustrated in the FACS profiles (B). These sorted cells were further stimulated with α-GalCer in the presence of irradiated macrophages from Jα18−/− mice as APCs. IL-17 was measured in the supernatants 3 days later. No cytokines were detected in the absence of α-GalCer or APCs and stimulated APCs were also negative (data not shown). Data represent the mean ± SD of 4 individual experiments.
Fig. 2.
Fig. 2.
Characterization of IL-17-producing thymic iNKT cells. (A) Intracellular IL-17 staining was performed after in vitro stimulation of enriched thymic iNKT cells with PMA+ionomycin for 4 h in the presence of brefeldin A. Representative FACS profile of CD44 and IL-17 expression by gated CD1d tetramerposCD8neg St1 (CD44negNK1.1neg), St2, CD4pos (CD44posNK1.1negCD4pos), St2, CD4neg (CD44posNK1.1negCD4neg), or St3 (CD44posNK1.1pos). (B) Thymic lobes were collected from Jα18−/− fetuses at day 17 of gestation. Thymic iNKT cells corresponding to the 4 differentiation stages were electronically sorted and added to the thymic lobes until day 14 when they were recovered. Representative FACS profile of CD4 and NK1.1 expression (Lower) among gated CD1d tetramerposCD44pos cells (Upper). Data are representative of 3 independent experiments. Percentages of each subset are indicated in quadrants.
Fig. 3.
Fig. 3.
ROR-γt mRNA expression in thymic iNKT cell subsets. Histograms represent the relative mRNA expression in the 4 distinct subsets from thymic CD1d tetramerposCD8neg cells, namely CD44negNK1.1neg (St1), CD44posNK1.1negCD4pos (St2, CD4pos), CD44posNK1.1negCD4neg (St2, CD4neg), and CD44posNK1.1pos (St3), processed immediately for RNA extraction after sorting. The mRNA expression profile was assessed by a PCR array (SuperArray Bioscience Corporation) and data represent relative values established by comparison with 4 housekeeping genes: Hprt, Gusb, Hsp90, and Gadph (only the latter are represented in the figure).
Fig. 4.
Fig. 4.
Characterization of thymic iNKT ROR-γtpos cells. (A and B) Expression of ROR-γt among gated CD1d tetramerpos CD8pos or CD8neg (A Left) or CD1d tetramerpos CD24pos or CD24neg (B Left) iNKT cells using Rorc(γt)-GfpTG mice. (A Right and B Right) Negative controls with empty tetramers. (C Left) CD4, CD44, and NK1.1 expression was analyzed among gated CD1d tetramerpos CD8neg CD24neg ROR-γtpos or ROR-γtneg iNKT cells. (C Right) Negative control with FITC channel in wild-type mice. Data are representative of 3 independent experiments. Percentages of each subset are indicated in quadrants.
Fig. 5.
Fig. 5.
The thymic iNKT CD44posNK1.1negCD4neg ROR-γtpos subset is the major source of IL-17. (A) Intracellular IL-17 staining was performed after in vitro stimulation of the 4 sorted thymic iNKT cell populations, namely CD44negNK1.1neg (St1), CD44posNK1.1negCD4pos (St2, CD4pos), CD44posNK1.1negCD4neg (St2, CD4neg), and CD44posNK1.1pos (St3) cells, with PMA+ionomycin during 4 h in the presence of brefeldin A. Numbers in quadrants indicate percentages of each subset. (B) Thymic iNKT St2, CD4pos and St2, CD4neg were sorted into ROR-γtpos or ROR-γtneg fractions and further stimulated with α-GalCer in the presence of irradiated macrophages from Jα18−/− mice as APCs. IL-17 was measured in the supernatants 3 days later. Data are representative of 3 independent experiments.
Fig. 6.
Fig. 6.
Analysis of ROR-γt expression and IL-17 production by iNKT splenocytes. (A) CD1d tetramerpos splenic iNKT cells were gated from fluorescent cells recovered from Rorc(γt)-GfpTG mice. The expression of CD4 and NK1.1 was analyzed among gated CD1d tetramerpos ROR-γtneg or ROR-γtpos subsets. (B) CD1d tetramerpos iNKT splenocytes were sorted into ROR-γtpos or ROR-γtneg fractions and further stimulated with α-GalCer in the presence of irradiated macrophages from Jα18−/− mice as APCs. IL-17 was measured in the supernatants 3 days later. Data are representative of 2 independent experiments.
Fig. 7.
Fig. 7.
ROR-γt is necessary for IL-17 production by iNKT cells. Total thymocytes from Jα18−/− mice were cultured in the presence of IL-7 alone (A) or together with thymic CD1d tetramerposCD8neg iNKT cells from Rorc(γt)-GfpTG mice sorted into St1 (CD44negNK1.1neg) ROR-γtneg (B), St2 (CD44posNK1.1neg) CD4neg ROR-γtneg (C), St2 (CD44posNK1.1neg) CD4pos ROR-γtneg (D), or ROR-γtpos subsets (E and F). Percentages of CD1d tetramerpos ROR-γtpos and ROR-γtneg cells generated within 4 days are indicated in the first column. (B–F) The expression of NK1.1, CD4, and CD44 was analyzed among gated CD1d tetramerpos ROR-γtneg (B–D and F) or ROR-γtpos populations (E). A fraction of the cells recovered after culture was further stimulated with PMA+ionomycin for 4 h in the presence of brefeldin A. (B–F) The percentage of IL-17pos cells was then analyzed among gated CD1d tetramerpos ROR-γtneg (B–D) or ROR-γtpos (E) cells as shown in the last column. Numbers in quadrants indicate the respective percentages. Data are representative of 2 independent experiments.
Fig. 8.
Fig. 8.
Proposed model for IL-17-producing iNKT cell differentiation. The majority of immature CD4posCD8posCD24posROR-γtpos iNKT cell precursors will lose ROR-γt expression when they differentiate into IL-4 and IFN-γ producers (classical pathway) whereas the minor fraction that remains ROR-γtpos will give rise to IL-17-producing iNKT cells (alternative pathway).
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