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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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Lymphocyte responses induced against GD3 in immunized mice produce IL-4. 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. Control mice (Freund's adjuvant only) were injected with CFA on day 0 followed by IFA alone on day 7. (a) Splenocytes were collected on day 14 and stimulated with irradiated syngeneic APCs loaded with GD3, GM2, or without ganglioside (None) as indicated. IL-4 and IFN-γ production were detected by ELISPOT assay. (b) In other sets of mice, hepatic mononuclear cells and thymocytes were tested for IL-4 secretion. Data represent mean ± SEM of triplicate wells from four mice and similar results were observed in repeated experiments.
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fig1: Lymphocyte responses induced against GD3 in immunized mice produce IL-4. 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. Control mice (Freund's adjuvant only) were injected with CFA on day 0 followed by IFA alone on day 7. (a) Splenocytes were collected on day 14 and stimulated with irradiated syngeneic APCs loaded with GD3, GM2, or without ganglioside (None) as indicated. IL-4 and IFN-γ production were detected by ELISPOT assay. (b) In other sets of mice, hepatic mononuclear cells and thymocytes were tested for IL-4 secretion. Data represent mean ± SEM of triplicate wells from four mice and similar results were observed in repeated experiments.

Mentions: Immunization of mice with human melanoma SK-MEL-28 cells (GD3+, GM2−) expanded a population of splenocytes that produced IL-4 in response to GD3-loaded APCs but did not produce detectable IFN-γ after two immunizations (Fig. 1 a). These GD3-reactive splenocytes were not detectable in the spleens of unimmunized mice, which demonstrated that immunization with the GD3+ melanoma was critical for this response. Reactivity to GD3 was not due to a general, nonspecific reactivity because splenocytes from immunized mice did not recognize unloaded APCs or APCs loaded with GM2, a ganglioside not expressed by SK-MEL-28. This indicates that these splenocytes from immunized mice have some degree of specificity. To exclude the possibility that the splenocyte response against GD3 was induced by lipid components in Freund's adjuvant, we tested splenocytes from mice injected with Freund's adjuvant only. Splenocytes from these mice showed no significant reactivity to unloaded or GD3-loaded APCs, indicating that the splenocyte response to GD3 observed in SK-MEL-28–immunized mice was not due to the Freund's adjuvant. In immunized mice, GD3-reactive lymphocytes were also detected in the liver but not in the thymus (Fig. 1 b).


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

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

Lymphocyte responses induced against GD3 in immunized mice produce IL-4. 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. Control mice (Freund's adjuvant only) were injected with CFA on day 0 followed by IFA alone on day 7. (a) Splenocytes were collected on day 14 and stimulated with irradiated syngeneic APCs loaded with GD3, GM2, or without ganglioside (None) as indicated. IL-4 and IFN-γ production were detected by ELISPOT assay. (b) In other sets of mice, hepatic mononuclear cells and thymocytes were tested for IL-4 secretion. Data represent mean ± SEM of triplicate wells from four mice and similar results were observed in repeated experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196074&req=5

fig1: Lymphocyte responses induced against GD3 in immunized mice produce IL-4. 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. Control mice (Freund's adjuvant only) were injected with CFA on day 0 followed by IFA alone on day 7. (a) Splenocytes were collected on day 14 and stimulated with irradiated syngeneic APCs loaded with GD3, GM2, or without ganglioside (None) as indicated. IL-4 and IFN-γ production were detected by ELISPOT assay. (b) In other sets of mice, hepatic mononuclear cells and thymocytes were tested for IL-4 secretion. Data represent mean ± SEM of triplicate wells from four mice and similar results were observed in repeated experiments.
Mentions: Immunization of mice with human melanoma SK-MEL-28 cells (GD3+, GM2−) expanded a population of splenocytes that produced IL-4 in response to GD3-loaded APCs but did not produce detectable IFN-γ after two immunizations (Fig. 1 a). These GD3-reactive splenocytes were not detectable in the spleens of unimmunized mice, which demonstrated that immunization with the GD3+ melanoma was critical for this response. Reactivity to GD3 was not due to a general, nonspecific reactivity because splenocytes from immunized mice did not recognize unloaded APCs or APCs loaded with GM2, a ganglioside not expressed by SK-MEL-28. This indicates that these splenocytes from immunized mice have some degree of specificity. To exclude the possibility that the splenocyte response against GD3 was induced by lipid components in Freund's adjuvant, we tested splenocytes from mice injected with Freund's adjuvant only. Splenocytes from these mice showed no significant reactivity to unloaded or GD3-loaded APCs, indicating that the splenocyte response to GD3 observed in SK-MEL-28–immunized mice was not due to the Freund's adjuvant. In immunized mice, GD3-reactive lymphocytes were also detected in the liver but not in the thymus (Fig. 1 b).

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