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Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation.

Im JS, Arora P, Bricard G, Molano A, Venkataswamy MM, Baine I, Jerud ES, Goldberg MF, Baena A, Yu KO, Ndonye RM, Howell AR, Yuan W, Cresswell P, Chang YT, Illarionov PA, Besra GS, Porcelli SA - Immunity (2009)

Bottom Line: We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation.Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins.Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology , Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

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Functional Consequences of Membrane Lipid Raft Association of Glycolipid-Loaded CD1d Molecules(A) C57BL/6 BMDCs were pulsed for 18 hr with the indicated glycolipids, washed, and incubated for 15 min in medium alone (hatched bars) or medium containing 10 mM MβCD (solid bars). Cells were then fixed (1% paraformaldehyde) and used for stimulating autologous splenocytes for 24 hr, after which supernatant concentrations of IFN-γ and IL-4 were measured. Dotted lines indicate the background levels of cytokines in cultures with BMDC pulsed with vehicle only. Means and standard deviations for triplicate cultures are shown, and results are representative of three experiments. ∗∗p < 0.01 (ANOVA, Bonferroni post-test).(B) Effect of MβCD on in vivo presentation of αGalCer analogs. Splenic DCs were pulsed ex vivo with the indicated glycolipids for 18 hr and then incubated for 15 min in medium without (−, hatched bars) or with 10 mM MβCD (+, solid bars). After extensive washing, the cells were injected i.p. into naive C57BL/6 mice (106 cells/mouse). Serum was assayed for IL-4 at 2 hr and for IFN-γ at 24 hr postinjection. Data shown are means and standard deviations for pooled data from two experiments that had three mice per group. ∗∗p < 0.01 (ANOVA, Bonferroni post-test); NS, not significant (p > 0.05).
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fig7: Functional Consequences of Membrane Lipid Raft Association of Glycolipid-Loaded CD1d Molecules(A) C57BL/6 BMDCs were pulsed for 18 hr with the indicated glycolipids, washed, and incubated for 15 min in medium alone (hatched bars) or medium containing 10 mM MβCD (solid bars). Cells were then fixed (1% paraformaldehyde) and used for stimulating autologous splenocytes for 24 hr, after which supernatant concentrations of IFN-γ and IL-4 were measured. Dotted lines indicate the background levels of cytokines in cultures with BMDC pulsed with vehicle only. Means and standard deviations for triplicate cultures are shown, and results are representative of three experiments. ∗∗p < 0.01 (ANOVA, Bonferroni post-test).(B) Effect of MβCD on in vivo presentation of αGalCer analogs. Splenic DCs were pulsed ex vivo with the indicated glycolipids for 18 hr and then incubated for 15 min in medium without (−, hatched bars) or with 10 mM MβCD (+, solid bars). After extensive washing, the cells were injected i.p. into naive C57BL/6 mice (106 cells/mouse). Serum was assayed for IL-4 at 2 hr and for IFN-γ at 24 hr postinjection. Data shown are means and standard deviations for pooled data from two experiments that had three mice per group. ∗∗p < 0.01 (ANOVA, Bonferroni post-test); NS, not significant (p > 0.05).

Mentions: These results strongly indicated that αGalCer analogs differ profoundly with respect to presentation by lipid-raft-associated or nonraft-associated mCD1d molecules, and such a difference can be envisioned to have major effects on the strength and quality of iNKT cell activation (Park et al., 2005). To assess functional consequences of raft localization of CD1d-glycolipid complexes, we analyzed the effects of methyl-β-cyclodextrin (MβCD) treatment on the iNKT cell stimulatory capacity of CD1d-glycolipid complexes in vitro and in vivo (Figure 7). MβCD substantially disrupts lipid raft microdomain structures by extracting cholesterol from the outer leaflet of the plasma membrane and reduces the raft association of plasma membrane CD1d molecules (Park et al., 2005). Treatment of DCs pulsed with αGalCer-C26:0 with MβCD markedly reduced their ability to stimulate IFN-γ production by splenocytes and had no effect on stimulation of IL-4. This effect was not observed for in vitro stimulations with αGalCer-C20:2- or αGalCer-C10:0-pulsed DCs, which were unaffected by MβCD (Figure 7A). A similar result was obtained for in vivo iNKT cell stimulation after injection of MβCD-treated DCs (Figure 7B). These results implied that cholesterol-rich plasma membrane lipid rafts were highly relevant for the mixed cytokine response to αGalCer-C26:0 but were dispensable for iNKT cell responses to Th2 cell-type cytokine-biasing agonists, consistent with their lack of presentation by lipid-raft-associated CD1d molecules.


Kinetics and cellular site of glycolipid loading control the outcome of natural killer T cell activation.

Im JS, Arora P, Bricard G, Molano A, Venkataswamy MM, Baine I, Jerud ES, Goldberg MF, Baena A, Yu KO, Ndonye RM, Howell AR, Yuan W, Cresswell P, Chang YT, Illarionov PA, Besra GS, Porcelli SA - Immunity (2009)

Functional Consequences of Membrane Lipid Raft Association of Glycolipid-Loaded CD1d Molecules(A) C57BL/6 BMDCs were pulsed for 18 hr with the indicated glycolipids, washed, and incubated for 15 min in medium alone (hatched bars) or medium containing 10 mM MβCD (solid bars). Cells were then fixed (1% paraformaldehyde) and used for stimulating autologous splenocytes for 24 hr, after which supernatant concentrations of IFN-γ and IL-4 were measured. Dotted lines indicate the background levels of cytokines in cultures with BMDC pulsed with vehicle only. Means and standard deviations for triplicate cultures are shown, and results are representative of three experiments. ∗∗p < 0.01 (ANOVA, Bonferroni post-test).(B) Effect of MβCD on in vivo presentation of αGalCer analogs. Splenic DCs were pulsed ex vivo with the indicated glycolipids for 18 hr and then incubated for 15 min in medium without (−, hatched bars) or with 10 mM MβCD (+, solid bars). After extensive washing, the cells were injected i.p. into naive C57BL/6 mice (106 cells/mouse). Serum was assayed for IL-4 at 2 hr and for IFN-γ at 24 hr postinjection. Data shown are means and standard deviations for pooled data from two experiments that had three mice per group. ∗∗p < 0.01 (ANOVA, Bonferroni post-test); NS, not significant (p > 0.05).
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fig7: Functional Consequences of Membrane Lipid Raft Association of Glycolipid-Loaded CD1d Molecules(A) C57BL/6 BMDCs were pulsed for 18 hr with the indicated glycolipids, washed, and incubated for 15 min in medium alone (hatched bars) or medium containing 10 mM MβCD (solid bars). Cells were then fixed (1% paraformaldehyde) and used for stimulating autologous splenocytes for 24 hr, after which supernatant concentrations of IFN-γ and IL-4 were measured. Dotted lines indicate the background levels of cytokines in cultures with BMDC pulsed with vehicle only. Means and standard deviations for triplicate cultures are shown, and results are representative of three experiments. ∗∗p < 0.01 (ANOVA, Bonferroni post-test).(B) Effect of MβCD on in vivo presentation of αGalCer analogs. Splenic DCs were pulsed ex vivo with the indicated glycolipids for 18 hr and then incubated for 15 min in medium without (−, hatched bars) or with 10 mM MβCD (+, solid bars). After extensive washing, the cells were injected i.p. into naive C57BL/6 mice (106 cells/mouse). Serum was assayed for IL-4 at 2 hr and for IFN-γ at 24 hr postinjection. Data shown are means and standard deviations for pooled data from two experiments that had three mice per group. ∗∗p < 0.01 (ANOVA, Bonferroni post-test); NS, not significant (p > 0.05).
Mentions: These results strongly indicated that αGalCer analogs differ profoundly with respect to presentation by lipid-raft-associated or nonraft-associated mCD1d molecules, and such a difference can be envisioned to have major effects on the strength and quality of iNKT cell activation (Park et al., 2005). To assess functional consequences of raft localization of CD1d-glycolipid complexes, we analyzed the effects of methyl-β-cyclodextrin (MβCD) treatment on the iNKT cell stimulatory capacity of CD1d-glycolipid complexes in vitro and in vivo (Figure 7). MβCD substantially disrupts lipid raft microdomain structures by extracting cholesterol from the outer leaflet of the plasma membrane and reduces the raft association of plasma membrane CD1d molecules (Park et al., 2005). Treatment of DCs pulsed with αGalCer-C26:0 with MβCD markedly reduced their ability to stimulate IFN-γ production by splenocytes and had no effect on stimulation of IL-4. This effect was not observed for in vitro stimulations with αGalCer-C20:2- or αGalCer-C10:0-pulsed DCs, which were unaffected by MβCD (Figure 7A). A similar result was obtained for in vivo iNKT cell stimulation after injection of MβCD-treated DCs (Figure 7B). These results implied that cholesterol-rich plasma membrane lipid rafts were highly relevant for the mixed cytokine response to αGalCer-C26:0 but were dispensable for iNKT cell responses to Th2 cell-type cytokine-biasing agonists, consistent with their lack of presentation by lipid-raft-associated CD1d molecules.

Bottom Line: We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation.Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins.Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology , Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

Show MeSH
Related in: MedlinePlus