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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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GD3-reactive cells are CD4+8− NKT cells and CD4−8− NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0, and boosted with 2 × 106 SK-MEL-28 cells mixed with IFA on day 7. Splenocytes pooled from six to eight immunized mice were separated into subpopulations based on their surface expression of NK1.1, CD3, CD4, and CD8. Total cells (unseparated splenocytes), total NKT cells (CD3+ NK1.1+), total conventional T cells (CD3+ NK1.1−), CD4+ NKT cells (CD4+ CD8− CD3+ NK1.1+), CD8+ NKT cells (CD4− CD8+ CD3+ NK1.1+), DN NKT cells (CD4− CD8− CD3+ NK1.1+), and classic NK cells (CD3− NK1.1+) populations were stimulated with irradiated syngeneic APCs in the presence (+ columns, closed squares) or absence (− columns, open squares) of GD3. IL-4 production by each cell population was detected by ELISPOT assay. Horizontal lines indicate mean values.
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fig3: GD3-reactive cells are CD4+8− NKT cells and CD4−8− NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0, and boosted with 2 × 106 SK-MEL-28 cells mixed with IFA on day 7. Splenocytes pooled from six to eight immunized mice were separated into subpopulations based on their surface expression of NK1.1, CD3, CD4, and CD8. Total cells (unseparated splenocytes), total NKT cells (CD3+ NK1.1+), total conventional T cells (CD3+ NK1.1−), CD4+ NKT cells (CD4+ CD8− CD3+ NK1.1+), CD8+ NKT cells (CD4− CD8+ CD3+ NK1.1+), DN NKT cells (CD4− CD8− CD3+ NK1.1+), and classic NK cells (CD3− NK1.1+) populations were stimulated with irradiated syngeneic APCs in the presence (+ columns, closed squares) or absence (− columns, open squares) of GD3. IL-4 production by each cell population was detected by ELISPOT assay. Horizontal lines indicate mean values.

Mentions: Splenocytes from immunized mice were sorted based on CD3, NK1.1, CD4, and CD8 expression. When each splenocyte subpopulation was tested for reactivity against GD3-loaded APCs or unloaded APCs, GD3 reactivity was observed only among the NK1.1+, CD3+ subpopulations (Fig. 3). Two groups of GD3-reactive NKT cells were detected: CD4+CD8− and CD4−CD8− (DN). We did not detect CD8+ NKT cells reactive to GD3. There was no reactivity against GD3 among T cells (CD3+, NK1.1− lymphocytes) or classical NK cells (CD3−, NK1.1+ lymphocytes).


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

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

GD3-reactive cells are CD4+8− NKT cells and CD4−8− NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0, and boosted with 2 × 106 SK-MEL-28 cells mixed with IFA on day 7. Splenocytes pooled from six to eight immunized mice were separated into subpopulations based on their surface expression of NK1.1, CD3, CD4, and CD8. Total cells (unseparated splenocytes), total NKT cells (CD3+ NK1.1+), total conventional T cells (CD3+ NK1.1−), CD4+ NKT cells (CD4+ CD8− CD3+ NK1.1+), CD8+ NKT cells (CD4− CD8+ CD3+ NK1.1+), DN NKT cells (CD4− CD8− CD3+ NK1.1+), and classic NK cells (CD3− NK1.1+) populations were stimulated with irradiated syngeneic APCs in the presence (+ columns, closed squares) or absence (− columns, open squares) of GD3. IL-4 production by each cell population was detected by ELISPOT assay. Horizontal lines indicate mean values.
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Related In: Results  -  Collection

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

fig3: GD3-reactive cells are CD4+8− NKT cells and CD4−8− NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0, and boosted with 2 × 106 SK-MEL-28 cells mixed with IFA on day 7. Splenocytes pooled from six to eight immunized mice were separated into subpopulations based on their surface expression of NK1.1, CD3, CD4, and CD8. Total cells (unseparated splenocytes), total NKT cells (CD3+ NK1.1+), total conventional T cells (CD3+ NK1.1−), CD4+ NKT cells (CD4+ CD8− CD3+ NK1.1+), CD8+ NKT cells (CD4− CD8+ CD3+ NK1.1+), DN NKT cells (CD4− CD8− CD3+ NK1.1+), and classic NK cells (CD3− NK1.1+) populations were stimulated with irradiated syngeneic APCs in the presence (+ columns, closed squares) or absence (− columns, open squares) of GD3. IL-4 production by each cell population was detected by ELISPOT assay. Horizontal lines indicate mean values.
Mentions: Splenocytes from immunized mice were sorted based on CD3, NK1.1, CD4, and CD8 expression. When each splenocyte subpopulation was tested for reactivity against GD3-loaded APCs or unloaded APCs, GD3 reactivity was observed only among the NK1.1+, CD3+ subpopulations (Fig. 3). Two groups of GD3-reactive NKT cells were detected: CD4+CD8− and CD4−CD8− (DN). We did not detect CD8+ NKT cells reactive to GD3. There was no reactivity against GD3 among T cells (CD3+, NK1.1− lymphocytes) or classical NK cells (CD3−, NK1.1+ lymphocytes).

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