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. 2012 Mar 1;188(5):2118-26.
doi: 10.4049/jimmunol.1103342. Epub 2012 Jan 27.

MicroRNA miR-150 is involved in Vα14 invariant NKT cell development and function

Quanhui Zheng  1 , Li Zhou, Qing-Sheng Mi
Affiliations

MicroRNA miR-150 is involved in Vα14 invariant NKT cell development and function

Quanhui Zheng et al. J Immunol. .

Abstract

CD1d-restricted Vα14 invariant NKT (iNKT) cells play an important role in the regulation of diverse immune responses. MicroRNA-mediated RNA interference is emerging as a crucial regulatory mechanism in the control of iNKT cell differentiation and function. Yet, roles of specific microRNAs in the development and function of iNKT cells remain to be further addressed. In this study, we identified the gradually increased expression of microRNA-150 (miR-150) during the maturation of iNKT cells in thymus. Using miR-150 knockout (KO) mice, we found that miR-150 deletion resulted in an interruption of iNKT cell final maturation in both thymus and periphery. Upon activation, iNKT cells from miR-150KO mice showed significantly increased IFN-γ production compared with wild-type iNKT cells. Bone marrow-transferring experiments demonstrated the cell-intrinsic characteristics of iNKT cell maturation and functional defects in mice lacking miR-150. Furthermore, miR-150 target c-Myb was significantly upregulated in miR-150KO iNKT cells, which potentially contribute to iNKT cell defects in miR-150KO mice. Our data define a specific role of miR-150 in the development and function of iNKT cells.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest

Figures

FIGURE 1.
FIGURE 1.
Expression of miR-150 is up-regulated during the development and activation of iNKT cells. A, Different developmental stages (CD44−NK1.1−, CD44+NK1.1− and CD44+NK1.1+) of thymic iNKT cells were sorted from C57BL/6 mice. B, Taqman real-time PCR analysis of miR-150 transcript in CD44−NK1.1−, CD44+NK1.1− and CD44+NK1.1+ iNKT cells from the thymuses of C57BL/6 mice. Results were the mean of triplicate and normalized to a control gene (snoRNU 202). Error bars are SD. * P <0.05; ** P <0.01. Results are representatives of three independent experiments. C, C57BL/6 mice were stimulated in vivo with α -GalCer for 3 days, spleen iNKT cells were sorted and RNA was extracted. MiR-150 expression was detected by Taqman real-time PCR. Results were the mean of triplicate and normalized to a control gene (snoRNU 202). Error bars are SD. Results are representatives of three independent experiments.
FIGURE 2.
FIGURE 2.
Defective iNKT cell development in miR-150KO mice. A, Representative dot plots of thymus iNKT cells stained by anti- TCRβ Ab and CD1d-Tetramer from miR-150KO and WT mice. B and C, The frequency (B) and number(C) of TCRβ+CD1d-Tetramer+ iNKT cells in the thymus of miR-150KO and WT mice. Each point represents one individual mouse and the mean values are indicated by the middle horizontal lines from 3–5 independent experiments (3–5 mice/experiment). D, The representative staining of iNKT cells (gated on B220 negative cells) in lymph nodes, spleen, bone marrow, and liver from miR-150KO and WT mice. E and F, The frequency (E) and cell number (F) of iNKT cells in the lymph nodes, spleen, bone marrow and liver of miR-150KO and WT mice, Each point represents one individual mouse and the mean values are indicated by middle horizontal lines from 3–5 independent experiments (3–5 mice/experiment). Statistical analysis was performed with Prism 5.0 (GraphPad Software). Differences were considered statistically significant when P < 0.05.
FIGURE 3.
FIGURE 3.
Defective iNKT cell maturation in miR-150KO mice. A, Dot plots depict CD44 and NK1.1 expression in thymus iNKT cells of miR-150KO and WT control mice. B-C, events showed the percentage (B) and number (C) of iNKT cell sub-populations based on their CD44 and NK1.1 expression patterns gated on TCRβ and CD1d-Tetramer double-positive iNKT cells in the thymus. D, Dot plots depict CD122, CD69, ly49C and T-bet expression in the thymus iNKT cells of miR-150KO and WT control mice. E, Percentage of CD122, CD69, Ly49c and T-bet positive cells gated on TCRβ and CD1d-Tetramer double-positive iNKT cells in the thymus iNKT cells of miR-150KO and WT control mice. Each point represents one individual mouse and the mean values are indicated by the middle horizontal lines from 3–5 independent experiments (3–5 mice/experiment). F, Representative dot plots of NK1.1 staining (gated on B200 negative iNKT cells) in lymph nodes, spleen, bone marrow and liver of miR-150KO and WT control mice. G, Percentage of NK1.1 positive cells gated on lymph nodes, spleen, bone marrow and liver of miR-150KO and WT control mice Each point represents one individual mouse and the mean values are indicated by horizontal lines from 3–5 independent experiments (3–5 mice/experiment).
FIGURE 4.
FIGURE 4.
MiR-150 contributes to thymus iNKT cell survival during iNKT cell development. A, Histogram plots depict CD1d expression in CD4+CD8+ (DP) thymocytes of WT control (filled histogram) and miR-150KO mice (open histogram). Data are representative of three independent experiments (3–5 mice/experiment). B, WT control and miR-150KO mice were injected with BrdU, 24 hours later, thymocytes were isolated and stained for TCRβ, CD1d-Tetramer, and BrdU Abs. BrdU positive cells in gated TCRβ+-Tetramer+ double positive (iNKT) cells are shown. C, Percentage of BrdU positive cells in the thymus iNKT cells of WT control and miR-150 KO mice are shown. Results are representatives of three independent experiments (3–5 mice/experiment). D, Thymocytes were stained for TCRβ, CD1d-Tetramer, and Annexin V. Gated TCRβ+ -Tetramer+ double positive (iNKT) cells are shown. E, Percentage of Annexin V positive cells in gated thymus iNKT cells of WT control and miR-150KO mice. Data are representative of two independent experiments (3–5 mice/experiment).
FIGURE 5.
FIGURE 5.
Increased α-GalCer-dependent iNKT cell function in the miR-150 KO iNKT cells. A, Intracellular staining analysis of IL-4, IFN-γ and CD69 expression from spleen iNKT cells of WT control and miR-150KOmice after in vivo α-GalCer stimulation for 2 hours. B, Percentage of IL-4 and IFN-γ positive iNKT cells, and CD69 positive iNKT cells in the WT and miR-150KO mice after in vivo α-GalCer stimulation for 2 hours. Results are representative of 3 independent experiments (3–5 mice/experiment). C, Whole splenocytes from WT control and miR-150KO mice were treated with PMA and Ionomycin in vitro for 3 hours. IL-4 and INF-γ expression in spleen iNKT cells were analyzed by intracellular staining. D, Percentage of IL-4 and IFN-γ positive iNKT cells in the WT control and miR-150KOmice after PMA and Ionomycin treatment in vitro for 3 hours are shown. Results are representative of 3–5 independent experiments (3–5 mice/experiment).
FIGURE 6.
FIGURE 6.
Defective iNKT cell development but increased iNKT cell function is cell intrinsic in miR-150KO mice. CD45.2+ miR-150KO and CD45.1+ B6.SJL bone marrows were mixed at a 1:1 ratio and transferred into lethally irradiated B6.SJL hosts. Eight weeks later, reconstituted animals were analyzed by flow cytometry. A, Frequency of thymus iNKT cells originated from the CD45.1+ B6.SJL bone marrows and CD45.2+ miR-150KO bone marrows. B, Flow cytometry analysis of iNKT developmental subsets gated from the B6.SJL and miR-150KO derived iNKT cells. C, Percentage of each iNKT developmental subset in the total iNKT cells of B6.SJL and miR-150KO mice are shown. D, Flow cytometry analysis of CD122 expression in the B6.SJL and miR-150KO derived iNKT cells. E, Percentage of CD122 positive cell in the B6.SJL and miR-150KO derived iNKT cells. F, Intracellular staining analysis of IL-4 and IFNγ expression from B6.SJL and miR-150KO derived spleen iNKT cells after in vivo α-GalCer stimulation for 2 hours. Data are representative of two independent experiments with the same trend (3–4 mice/experiment).
FIGURE 7.
FIGURE 7.
Up-regulation of c-Myb expression in the thymus iNKT cells of miR-150KO mice. Different stage of iNKT cells were sorted from the thymus of miR-150KO and WT control mice according to their CD44 and NK1.1 expression. To get enough cells for RNA analysis, stage 1 and stage 2 iNKT cells were combined as immature iNKT cells and stage 3 iNKT cells as mature iNKT cells. RNA was extracted from immature and mature iNKT cells, and c-Myb expression was analyzed by Taqman real-time PCR. Results were the representative of two independent experiments.
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