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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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Cytokine production by GD3-reactive NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0 without further boost (a), or 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 and day 14 (b). Splenocytes were collected at the time points indicated. Intracellular production of IL-4 (▪), IL-5 (▵), IL-10 (♦), IL-12 (○) and IFN-γ (▿) by NK1.1+ CD3+ cells was measured gating on CD3+ cells. Arrows indicate time of immunization. Data represent mean ± SEM from four mice, and similar results were observed in repeated experiments.
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fig8: Cytokine production by GD3-reactive NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0 without further boost (a), or 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 and day 14 (b). Splenocytes were collected at the time points indicated. Intracellular production of IL-4 (▪), IL-5 (▵), IL-10 (♦), IL-12 (○) and IFN-γ (▿) by NK1.1+ CD3+ cells was measured gating on CD3+ cells. Arrows indicate time of immunization. Data represent mean ± SEM from four mice, and similar results were observed in repeated experiments.

Mentions: The kinetics of cytokine production by GD3-reactive NKT cells was determined by intracellular cytokine FACS® analysis after immunizing mice weekly for up to three immunizations. After a single immunization on day 0, we detected production of IL-4, IL-10, and IFN-γ, which peaked on day 7 and diminished to baseline by day 10 (IL-4 and IFN-γ) or by day 17 (IL-10; Fig. 8 a). We did not detect IL-5 or IL-12 production by GD3-reactive NKT cells. These results indicate that, after a single immunization, the NKT cell response against GD3 is limited. In mice receiving weekly immunizations (Fig. 8 b), IFN-γ production by NKT cells was not sustained beyond day 10, despite booster immunizations on day 7 and day 14. This is consistent with our previous observations (Fig. 1 a), in which little IFN-γ secretion was detected after two immunizations and indicates that the IFN-γ response in these cells is transient despite further immunizations. IL-4 production by GD3-reactive NKT cells was detectable after the first immunization and peaked after the second immunization. However, despite a third immunization on day 14, IL-4 production decreased, indicating that the NKT cell response against GD3 could not be sustained. No IL-4 production was detected if splenocytes were incubated with unloaded APCs or if splenocytes from mice immunized with adjuvant alone were used (unpublished data), confirming the specificity of the response.


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

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

Cytokine production by GD3-reactive NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0 without further boost (a), or 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 and day 14 (b). Splenocytes were collected at the time points indicated. Intracellular production of IL-4 (▪), IL-5 (▵), IL-10 (♦), IL-12 (○) and IFN-γ (▿) by NK1.1+ CD3+ cells was measured gating on CD3+ cells. Arrows indicate time of immunization. Data represent mean ± SEM 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

fig8: Cytokine production by GD3-reactive NKTs. Mice were immunized with 2 × 106 SK-MEL-28 cells mixed with CFA on day 0 without further boost (a), or 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 and day 14 (b). Splenocytes were collected at the time points indicated. Intracellular production of IL-4 (▪), IL-5 (▵), IL-10 (♦), IL-12 (○) and IFN-γ (▿) by NK1.1+ CD3+ cells was measured gating on CD3+ cells. Arrows indicate time of immunization. Data represent mean ± SEM from four mice, and similar results were observed in repeated experiments.
Mentions: The kinetics of cytokine production by GD3-reactive NKT cells was determined by intracellular cytokine FACS® analysis after immunizing mice weekly for up to three immunizations. After a single immunization on day 0, we detected production of IL-4, IL-10, and IFN-γ, which peaked on day 7 and diminished to baseline by day 10 (IL-4 and IFN-γ) or by day 17 (IL-10; Fig. 8 a). We did not detect IL-5 or IL-12 production by GD3-reactive NKT cells. These results indicate that, after a single immunization, the NKT cell response against GD3 is limited. In mice receiving weekly immunizations (Fig. 8 b), IFN-γ production by NKT cells was not sustained beyond day 10, despite booster immunizations on day 7 and day 14. This is consistent with our previous observations (Fig. 1 a), in which little IFN-γ secretion was detected after two immunizations and indicates that the IFN-γ response in these cells is transient despite further immunizations. IL-4 production by GD3-reactive NKT cells was detectable after the first immunization and peaked after the second immunization. However, despite a third immunization on day 14, IL-4 production decreased, indicating that the NKT cell response against GD3 could not be sustained. No IL-4 production was detected if splenocytes were incubated with unloaded APCs or if splenocytes from mice immunized with adjuvant alone were used (unpublished data), confirming the specificity of the response.

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