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Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor.

Shiratori I, Ogasawara K, Saito T, Lanier LL, Arase H - J. Exp. Med. (2004)

Bottom Line: Transcripts of PILR ligand are present in many tissues, including some T cell lines.Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta.Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuoku, 260-8670, Japan.

ABSTRACT
Paired receptors that consist of highly related activating and inhibitory receptors are widely involved in the regulation of the immune system. Here, we report a mouse orthologue of the human activating paired immunoglobulin-like type 2 receptor (PILR) beta, which was cloned from a cDNA library of natural killer (NK) cells based on its ability to associate with the DAP12 signaling adaptor protein. The activating PILRbeta was expressed not only on NK cells but also on dendritic cells and macrophages. Furthermore, we have identified a novel CD99-like molecule as a ligand for the activating PILRbeta and inhibitory PILRalpha receptors. Transcripts of PILR ligand are present in many tissues, including some T cell lines. Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta. Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

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(A) Role of DAP12 in the cell surface expression and signal transduction of PILRβ, FLAG-PILRβ (solid line), and FLAG-PILRα (dashed line) was transfected into the MA5.8 cell line. Parental MA5.8 (dotted line) and the transfectants were stained with anti-FLAG mAb. Mean fluorescence intensities of anti-FLAG mAb staining for parental MA5.8, FLAG-PILRα, and FLAG-PILRβ–transfected cells were 2.93, 27.0, and 369.1, respectively. (B) DAP12, CD3ζ, or FcRγ were transfected into MA5.8-expressing FLAG-PILRβ using the pMx-IRES-GFP retrovirus vector. Transfectants were stained with anti-FLAG mAb and expression of FLAG on GFP-expressing cells is shown (solid line). FLAG expression on a mock transfectant was overlaid in this figure (dotted line). Mean fluorescence intensities of anti-FLAG mAb staining for FLAG-PILRβ–expressing cells transfected with DAP12, CD3ζ, and FcRγ were 1137.7, 56.4, and 33.9, respectively. (C) IL-2 production by MA5.8 cells and MA5.8 transfected with FLAG-PILRβ and DAP12, CD3ζ, or FcRγ and cultured for 1 d in the presence of immobilized anti-FLAG mAb (shaded bar) or control mAb (unshaded bar). IL-2 in the culture supernatants was measured by ELISA.
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fig2: (A) Role of DAP12 in the cell surface expression and signal transduction of PILRβ, FLAG-PILRβ (solid line), and FLAG-PILRα (dashed line) was transfected into the MA5.8 cell line. Parental MA5.8 (dotted line) and the transfectants were stained with anti-FLAG mAb. Mean fluorescence intensities of anti-FLAG mAb staining for parental MA5.8, FLAG-PILRα, and FLAG-PILRβ–transfected cells were 2.93, 27.0, and 369.1, respectively. (B) DAP12, CD3ζ, or FcRγ were transfected into MA5.8-expressing FLAG-PILRβ using the pMx-IRES-GFP retrovirus vector. Transfectants were stained with anti-FLAG mAb and expression of FLAG on GFP-expressing cells is shown (solid line). FLAG expression on a mock transfectant was overlaid in this figure (dotted line). Mean fluorescence intensities of anti-FLAG mAb staining for FLAG-PILRβ–expressing cells transfected with DAP12, CD3ζ, and FcRγ were 1137.7, 56.4, and 33.9, respectively. (C) IL-2 production by MA5.8 cells and MA5.8 transfected with FLAG-PILRβ and DAP12, CD3ζ, or FcRγ and cultured for 1 d in the presence of immobilized anti-FLAG mAb (shaded bar) or control mAb (unshaded bar). IL-2 in the culture supernatants was measured by ELISA.

Mentions: We analyzed the role of ITAM-bearing adaptor proteins, including CD3ζ, FcRγ, and DAP12, on the cell surface expression and signal transduction of mouse PILRβ. NH2-terminal FLAG-tagged PILRα (FLAG-PILRα) or PILRβ (FLAG-PILRβ) was transfected into a T cell hybridoma, MA5.8, that lacks CD3ζ, FcRγ, and DAP12. As shown in Fig. 2 A, FLAG-PILRα and FLAG-PILRβ were expressed on the cell surface of the MA5.8 transfectants, as demonstrated by staining with an anti-FLAG mAb. Of note, FLAG-PILRα was expressed at a higher level on MA5.8 than PILRβ. Next, we cotransfected CD3ζ, FcRγ, or DAP12 into M5.8 cells expressing FLAG-PILRβ (Fig. 2 B). The expression level of FLAG-PILRβ was substantially increased when DAP12, but not CD3ζ or FcRγ, was cotransfected into the MA5.8 cells bearing FLAG-PILRβ. In contrast, DAP10 did not substantially up-regulate the expression of FLAG-PILRβ (unpublished data). This suggests that DAP12 associates with PILRβ and stabilizes the cell surface expression of PILRβ.


Activation of natural killer cells and dendritic cells upon recognition of a novel CD99-like ligand by paired immunoglobulin-like type 2 receptor.

Shiratori I, Ogasawara K, Saito T, Lanier LL, Arase H - J. Exp. Med. (2004)

(A) Role of DAP12 in the cell surface expression and signal transduction of PILRβ, FLAG-PILRβ (solid line), and FLAG-PILRα (dashed line) was transfected into the MA5.8 cell line. Parental MA5.8 (dotted line) and the transfectants were stained with anti-FLAG mAb. Mean fluorescence intensities of anti-FLAG mAb staining for parental MA5.8, FLAG-PILRα, and FLAG-PILRβ–transfected cells were 2.93, 27.0, and 369.1, respectively. (B) DAP12, CD3ζ, or FcRγ were transfected into MA5.8-expressing FLAG-PILRβ using the pMx-IRES-GFP retrovirus vector. Transfectants were stained with anti-FLAG mAb and expression of FLAG on GFP-expressing cells is shown (solid line). FLAG expression on a mock transfectant was overlaid in this figure (dotted line). Mean fluorescence intensities of anti-FLAG mAb staining for FLAG-PILRβ–expressing cells transfected with DAP12, CD3ζ, and FcRγ were 1137.7, 56.4, and 33.9, respectively. (C) IL-2 production by MA5.8 cells and MA5.8 transfected with FLAG-PILRβ and DAP12, CD3ζ, or FcRγ and cultured for 1 d in the presence of immobilized anti-FLAG mAb (shaded bar) or control mAb (unshaded bar). IL-2 in the culture supernatants was measured by ELISA.
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Related In: Results  -  Collection

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fig2: (A) Role of DAP12 in the cell surface expression and signal transduction of PILRβ, FLAG-PILRβ (solid line), and FLAG-PILRα (dashed line) was transfected into the MA5.8 cell line. Parental MA5.8 (dotted line) and the transfectants were stained with anti-FLAG mAb. Mean fluorescence intensities of anti-FLAG mAb staining for parental MA5.8, FLAG-PILRα, and FLAG-PILRβ–transfected cells were 2.93, 27.0, and 369.1, respectively. (B) DAP12, CD3ζ, or FcRγ were transfected into MA5.8-expressing FLAG-PILRβ using the pMx-IRES-GFP retrovirus vector. Transfectants were stained with anti-FLAG mAb and expression of FLAG on GFP-expressing cells is shown (solid line). FLAG expression on a mock transfectant was overlaid in this figure (dotted line). Mean fluorescence intensities of anti-FLAG mAb staining for FLAG-PILRβ–expressing cells transfected with DAP12, CD3ζ, and FcRγ were 1137.7, 56.4, and 33.9, respectively. (C) IL-2 production by MA5.8 cells and MA5.8 transfected with FLAG-PILRβ and DAP12, CD3ζ, or FcRγ and cultured for 1 d in the presence of immobilized anti-FLAG mAb (shaded bar) or control mAb (unshaded bar). IL-2 in the culture supernatants was measured by ELISA.
Mentions: We analyzed the role of ITAM-bearing adaptor proteins, including CD3ζ, FcRγ, and DAP12, on the cell surface expression and signal transduction of mouse PILRβ. NH2-terminal FLAG-tagged PILRα (FLAG-PILRα) or PILRβ (FLAG-PILRβ) was transfected into a T cell hybridoma, MA5.8, that lacks CD3ζ, FcRγ, and DAP12. As shown in Fig. 2 A, FLAG-PILRα and FLAG-PILRβ were expressed on the cell surface of the MA5.8 transfectants, as demonstrated by staining with an anti-FLAG mAb. Of note, FLAG-PILRα was expressed at a higher level on MA5.8 than PILRβ. Next, we cotransfected CD3ζ, FcRγ, or DAP12 into M5.8 cells expressing FLAG-PILRβ (Fig. 2 B). The expression level of FLAG-PILRβ was substantially increased when DAP12, but not CD3ζ or FcRγ, was cotransfected into the MA5.8 cells bearing FLAG-PILRβ. In contrast, DAP10 did not substantially up-regulate the expression of FLAG-PILRβ (unpublished data). This suggests that DAP12 associates with PILRβ and stabilizes the cell surface expression of PILRβ.

Bottom Line: Transcripts of PILR ligand are present in many tissues, including some T cell lines.Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta.Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuoku, 260-8670, Japan.

ABSTRACT
Paired receptors that consist of highly related activating and inhibitory receptors are widely involved in the regulation of the immune system. Here, we report a mouse orthologue of the human activating paired immunoglobulin-like type 2 receptor (PILR) beta, which was cloned from a cDNA library of natural killer (NK) cells based on its ability to associate with the DAP12 signaling adaptor protein. The activating PILRbeta was expressed not only on NK cells but also on dendritic cells and macrophages. Furthermore, we have identified a novel CD99-like molecule as a ligand for the activating PILRbeta and inhibitory PILRalpha receptors. Transcripts of PILR ligand are present in many tissues, including some T cell lines. Cells expressing the PILR ligand specifically activated NK cells and dendritic cells that express the activating PILRbeta. Our findings reveal a new regulatory mechanism of innate immunity by PILR and its CD99-like ligand.

Show MeSH