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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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Lack of Detergent-like Cofactor Dependence of Th2 Cell-Type Cytokine-Biasing αGalCer Analogs(A) Glycolipids were incubated for 24 hr at 37°C with murine CD1d proteins in the absence (top row) or presence (bottom row) of 0.05% Triton X-100. The resulting complexes were assembled as tetramers and used for staining mouse iNKT hybridoma DN3A4.1-2. The dashed line histograms show background staining with unloaded tetramers, and the bold histograms show staining with glycolipid loaded tetramers.(B) The same analysis as in (A) except with human CD1d tetramers, which were also used for DN3A4.1-2 staining.(C) Glycolipids at various concentrations were incubated with immobilized mCD1d protein for 2 hr in pH 7.0 or pH 5.0 buffers with no additives (medium) or in the same buffers containing the indicated purified recombinant saposins (10 μg/ml) or Triton X-100 (0.05%). After washing, DN32.D3 iNKT hybridoma cells were added and supernatant concentrations of IL-2 were determined after an additional 14 hr of incubation. All results shown (A, B, and C) were representative of at least two independent experiments.
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fig5: Lack of Detergent-like Cofactor Dependence of Th2 Cell-Type Cytokine-Biasing αGalCer Analogs(A) Glycolipids were incubated for 24 hr at 37°C with murine CD1d proteins in the absence (top row) or presence (bottom row) of 0.05% Triton X-100. The resulting complexes were assembled as tetramers and used for staining mouse iNKT hybridoma DN3A4.1-2. The dashed line histograms show background staining with unloaded tetramers, and the bold histograms show staining with glycolipid loaded tetramers.(B) The same analysis as in (A) except with human CD1d tetramers, which were also used for DN3A4.1-2 staining.(C) Glycolipids at various concentrations were incubated with immobilized mCD1d protein for 2 hr in pH 7.0 or pH 5.0 buffers with no additives (medium) or in the same buffers containing the indicated purified recombinant saposins (10 μg/ml) or Triton X-100 (0.05%). After washing, DN32.D3 iNKT hybridoma cells were added and supernatant concentrations of IL-2 were determined after an additional 14 hr of incubation. All results shown (A, B, and C) were representative of at least two independent experiments.

Mentions: The efficient loading of CD1 molecules with lipids such as αGalCer-C26:0 is known to involve cofactors that enhance the transfer of lipids from micelles or membranes into the antigen-binding groove of the CD1 protein. The best known of these cofactors are resident intracellular lipid binding and transfer proteins including those belonging to the saposin family, GM2 activator proteins, and Niemann-Pick type C1 and C2 proteins (Zhou et al., 2004; Sagiv et al., 2006; Yuan et al., 2007; Schrantz et al., 2007). Most of these are known to have detergent-like activities that facilitate solubilization of lipids and their exchange between hydrophobic binding sites. In an in vitro model for this activity of endosomal lipid transfer proteins, we investigated the impact of detergent (0.05% Triton X-100) on the loading of αGalCer analogs onto purified mCD1d or hCD1d tetramers (Figures 5A and 5B). With mCD1d, there was a clear enhancing effect of Triton X-100 on the formation of tetramers that bound to iNKT cell TCRs when αGalCer-C26:0 or -C24:0 were used as ligands, whereas loading of all six Th2 cell-type cytokine-biasing analogs was optimal in the absence of detergent (Figure 5A). Largely similar findings were obtained with hCD1d tetramers (OCH failed to form hCD1d tetramers with iNKT cell-binding avidity under any conditions) (Figure 5B). Overall, this consistent lack of detergent dependence for loading of CD1d by all Th2 cell-type cytokine-biasing analogs was a further unifying feature of such compounds and pointed to differences in aqueous solubility of αGalCer analogs as an important determinant of their effects on iNKT cell activation.


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)

Lack of Detergent-like Cofactor Dependence of Th2 Cell-Type Cytokine-Biasing αGalCer Analogs(A) Glycolipids were incubated for 24 hr at 37°C with murine CD1d proteins in the absence (top row) or presence (bottom row) of 0.05% Triton X-100. The resulting complexes were assembled as tetramers and used for staining mouse iNKT hybridoma DN3A4.1-2. The dashed line histograms show background staining with unloaded tetramers, and the bold histograms show staining with glycolipid loaded tetramers.(B) The same analysis as in (A) except with human CD1d tetramers, which were also used for DN3A4.1-2 staining.(C) Glycolipids at various concentrations were incubated with immobilized mCD1d protein for 2 hr in pH 7.0 or pH 5.0 buffers with no additives (medium) or in the same buffers containing the indicated purified recombinant saposins (10 μg/ml) or Triton X-100 (0.05%). After washing, DN32.D3 iNKT hybridoma cells were added and supernatant concentrations of IL-2 were determined after an additional 14 hr of incubation. All results shown (A, B, and C) were representative of at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2719696&req=5

fig5: Lack of Detergent-like Cofactor Dependence of Th2 Cell-Type Cytokine-Biasing αGalCer Analogs(A) Glycolipids were incubated for 24 hr at 37°C with murine CD1d proteins in the absence (top row) or presence (bottom row) of 0.05% Triton X-100. The resulting complexes were assembled as tetramers and used for staining mouse iNKT hybridoma DN3A4.1-2. The dashed line histograms show background staining with unloaded tetramers, and the bold histograms show staining with glycolipid loaded tetramers.(B) The same analysis as in (A) except with human CD1d tetramers, which were also used for DN3A4.1-2 staining.(C) Glycolipids at various concentrations were incubated with immobilized mCD1d protein for 2 hr in pH 7.0 or pH 5.0 buffers with no additives (medium) or in the same buffers containing the indicated purified recombinant saposins (10 μg/ml) or Triton X-100 (0.05%). After washing, DN32.D3 iNKT hybridoma cells were added and supernatant concentrations of IL-2 were determined after an additional 14 hr of incubation. All results shown (A, B, and C) were representative of at least two independent experiments.
Mentions: The efficient loading of CD1 molecules with lipids such as αGalCer-C26:0 is known to involve cofactors that enhance the transfer of lipids from micelles or membranes into the antigen-binding groove of the CD1 protein. The best known of these cofactors are resident intracellular lipid binding and transfer proteins including those belonging to the saposin family, GM2 activator proteins, and Niemann-Pick type C1 and C2 proteins (Zhou et al., 2004; Sagiv et al., 2006; Yuan et al., 2007; Schrantz et al., 2007). Most of these are known to have detergent-like activities that facilitate solubilization of lipids and their exchange between hydrophobic binding sites. In an in vitro model for this activity of endosomal lipid transfer proteins, we investigated the impact of detergent (0.05% Triton X-100) on the loading of αGalCer analogs onto purified mCD1d or hCD1d tetramers (Figures 5A and 5B). With mCD1d, there was a clear enhancing effect of Triton X-100 on the formation of tetramers that bound to iNKT cell TCRs when αGalCer-C26:0 or -C24:0 were used as ligands, whereas loading of all six Th2 cell-type cytokine-biasing analogs was optimal in the absence of detergent (Figure 5A). Largely similar findings were obtained with hCD1d tetramers (OCH failed to form hCD1d tetramers with iNKT cell-binding avidity under any conditions) (Figure 5B). Overall, this consistent lack of detergent dependence for loading of CD1d by all Th2 cell-type cytokine-biasing analogs was a further unifying feature of such compounds and pointed to differences in aqueous solubility of αGalCer analogs as an important determinant of their effects on iNKT cell activation.

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