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Cross-presentation of disialoganglioside GD3 to natural killer T cells.

Wu DY, Segal NH, Sidobre S, Kronenberg M, Chapman PB - J. Exp. Med. (2003)

Bottom Line: GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10.Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo.This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

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Detection of GD3-reactive NKT cells using CD1d-GD3 tetramers. Splenocytes from mice immunized with SK-MEL-28 cells and Freund's adjuvant (immunized) or mice injected with Freund's adjuvant only (control) were analyzed by flow cytometry for binding to FITC-conjugated anti-CD3 mAb, PE-conjugated anti-NK1.1 mAb and TC-conjugated GD3-loaded mCD1d tetramers or unloaded mCD1d tetramers. Gating was performed on CD3+ cells. (a) Double immunofluorescence staining of cell surface expression of NK1.1 (PE, abscissa) and tetramer (TC, ordinate) from representative immunized and control mice. Splenocytes from an immunized mouse were stained with unloaded tetramer (right). Double-positive cells are indicated by the framed window. (b) Percent tetramer+ cells among total CD3+ NK1.1+ splenocytes from immunized and control mice stained with GD3-loaded CD1d tetramers, and immunized mice stained with unloaded tetramer. Each point represents a single mouse. The difference in the mean values (horizontal lines) was highly significant (P < 0.001, Kruskal-Wallis test).
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fig6: Detection of GD3-reactive NKT cells using CD1d-GD3 tetramers. Splenocytes from mice immunized with SK-MEL-28 cells and Freund's adjuvant (immunized) or mice injected with Freund's adjuvant only (control) were analyzed by flow cytometry for binding to FITC-conjugated anti-CD3 mAb, PE-conjugated anti-NK1.1 mAb and TC-conjugated GD3-loaded mCD1d tetramers or unloaded mCD1d tetramers. Gating was performed on CD3+ cells. (a) Double immunofluorescence staining of cell surface expression of NK1.1 (PE, abscissa) and tetramer (TC, ordinate) from representative immunized and control mice. Splenocytes from an immunized mouse were stained with unloaded tetramer (right). Double-positive cells are indicated by the framed window. (b) Percent tetramer+ cells among total CD3+ NK1.1+ splenocytes from immunized and control mice stained with GD3-loaded CD1d tetramers, and immunized mice stained with unloaded tetramer. Each point represents a single mouse. The difference in the mean values (horizontal lines) was highly significant (P < 0.001, Kruskal-Wallis test).

Mentions: We wished to confirm the NKT cell response against GD3 and quantitate it using a technique other than ELISPOT. To do this, we analyzed the NKT cell response against GD3 using GD3-loaded mouse CD1d tetramers. CD3+ NK1.1+ tetramer+ cells were detected among the splenocytes of immunized mice, but not among splenocytes of control mice injected only with Freund's adjuvant (Fig. 6 a, left and middle). As an important control, splenocytes from immunized mice stained with unloaded CD1d tetramers showed only background levels of tetramer staining, indicating the specificity of the GD3-loaded CD1d tetramers (Fig. 6 a, right). Overall, among the CD3+ NK1.1+ splenocytes in immunized mice, a mean of 4.5% tetramer+ NKT cells was compared with a mean of 1.2% in unimmunized mice or in immunized mice stained with unloaded CD1d tetramers, (P < 0.001; Fig. 6 b). These experiments confirmed that immunization expanded CD1d-restricted NKT cells recognizing GD3.


Cross-presentation of disialoganglioside GD3 to natural killer T cells.

Wu DY, Segal NH, Sidobre S, Kronenberg M, Chapman PB - J. Exp. Med. (2003)

Detection of GD3-reactive NKT cells using CD1d-GD3 tetramers. Splenocytes from mice immunized with SK-MEL-28 cells and Freund's adjuvant (immunized) or mice injected with Freund's adjuvant only (control) were analyzed by flow cytometry for binding to FITC-conjugated anti-CD3 mAb, PE-conjugated anti-NK1.1 mAb and TC-conjugated GD3-loaded mCD1d tetramers or unloaded mCD1d tetramers. Gating was performed on CD3+ cells. (a) Double immunofluorescence staining of cell surface expression of NK1.1 (PE, abscissa) and tetramer (TC, ordinate) from representative immunized and control mice. Splenocytes from an immunized mouse were stained with unloaded tetramer (right). Double-positive cells are indicated by the framed window. (b) Percent tetramer+ cells among total CD3+ NK1.1+ splenocytes from immunized and control mice stained with GD3-loaded CD1d tetramers, and immunized mice stained with unloaded tetramer. Each point represents a single mouse. The difference in the mean values (horizontal lines) was highly significant (P < 0.001, Kruskal-Wallis test).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196074&req=5

fig6: Detection of GD3-reactive NKT cells using CD1d-GD3 tetramers. Splenocytes from mice immunized with SK-MEL-28 cells and Freund's adjuvant (immunized) or mice injected with Freund's adjuvant only (control) were analyzed by flow cytometry for binding to FITC-conjugated anti-CD3 mAb, PE-conjugated anti-NK1.1 mAb and TC-conjugated GD3-loaded mCD1d tetramers or unloaded mCD1d tetramers. Gating was performed on CD3+ cells. (a) Double immunofluorescence staining of cell surface expression of NK1.1 (PE, abscissa) and tetramer (TC, ordinate) from representative immunized and control mice. Splenocytes from an immunized mouse were stained with unloaded tetramer (right). Double-positive cells are indicated by the framed window. (b) Percent tetramer+ cells among total CD3+ NK1.1+ splenocytes from immunized and control mice stained with GD3-loaded CD1d tetramers, and immunized mice stained with unloaded tetramer. Each point represents a single mouse. The difference in the mean values (horizontal lines) was highly significant (P < 0.001, Kruskal-Wallis test).
Mentions: We wished to confirm the NKT cell response against GD3 and quantitate it using a technique other than ELISPOT. To do this, we analyzed the NKT cell response against GD3 using GD3-loaded mouse CD1d tetramers. CD3+ NK1.1+ tetramer+ cells were detected among the splenocytes of immunized mice, but not among splenocytes of control mice injected only with Freund's adjuvant (Fig. 6 a, left and middle). As an important control, splenocytes from immunized mice stained with unloaded CD1d tetramers showed only background levels of tetramer staining, indicating the specificity of the GD3-loaded CD1d tetramers (Fig. 6 a, right). Overall, among the CD3+ NK1.1+ splenocytes in immunized mice, a mean of 4.5% tetramer+ NKT cells was compared with a mean of 1.2% in unimmunized mice or in immunized mice stained with unloaded CD1d tetramers, (P < 0.001; Fig. 6 b). These experiments confirmed that immunization expanded CD1d-restricted NKT cells recognizing GD3.

Bottom Line: GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10.Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo.This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

Show MeSH
Related in: MedlinePlus