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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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Related in: MedlinePlus

Cell Surface versus Intracellular Loading of mCD1d with Different αGalCer Analogs(A) On the left, surface accumulation of CD1d-αGalCer complexes was studied with JAWS II cells harvested after incubation with the indicated glycolipids for 1, 4, or 16 hr and then stained without permeabilization with mAb L363 (red). Cells were then fixed, stained with DAPI for nuclei visualization (blue), and analyzed by confocal microscopy. On the right, the intracellular formation and trafficking of CD1d-αGalCer complexes were analyzed by treatment of JAWS II cells with αGalCer analogs for 1, 4, and 16 hr followed by fixation and permeabilization. The cells were stained for CD1d-αGalCer complexes (L363, red), late endosomes and lysosomes (anti-LAMP-1, green), and nuclei (DAPI, blue). Results are representative of three separate experiments. The scale bar represents 10 μm.(B) Murine iNKT cell hybridoma cells (DN3A4.1-2) were stimulated with αGalCer analogs. As APCs for these stimulations, we used A20 cells transfected with wild-type CD1d (CD1d.WT) or cytoplasmic-tail-deleted CD1d (CD1d.TD). Supernatant concentrations of IL-2 release were determined after 12 hr. IL-2 production over a range of concentrations is shown for αGalCer-C26:0 and two analogs that produce a Th2 cell-type cytokine bias (αGalCer-C20:2 and -PGB1). The bar graph on the right shows relative potencies [(EC50 with presentation by CD1d.WT)/(EC50 with presentation by CD1d.TD)] of each glycolipid presented by CD1d.TD versus CD1d.WT.
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fig4: Cell Surface versus Intracellular Loading of mCD1d with Different αGalCer Analogs(A) On the left, surface accumulation of CD1d-αGalCer complexes was studied with JAWS II cells harvested after incubation with the indicated glycolipids for 1, 4, or 16 hr and then stained without permeabilization with mAb L363 (red). Cells were then fixed, stained with DAPI for nuclei visualization (blue), and analyzed by confocal microscopy. On the right, the intracellular formation and trafficking of CD1d-αGalCer complexes were analyzed by treatment of JAWS II cells with αGalCer analogs for 1, 4, and 16 hr followed by fixation and permeabilization. The cells were stained for CD1d-αGalCer complexes (L363, red), late endosomes and lysosomes (anti-LAMP-1, green), and nuclei (DAPI, blue). Results are representative of three separate experiments. The scale bar represents 10 μm.(B) Murine iNKT cell hybridoma cells (DN3A4.1-2) were stimulated with αGalCer analogs. As APCs for these stimulations, we used A20 cells transfected with wild-type CD1d (CD1d.WT) or cytoplasmic-tail-deleted CD1d (CD1d.TD). Supernatant concentrations of IL-2 release were determined after 12 hr. IL-2 production over a range of concentrations is shown for αGalCer-C26:0 and two analogs that produce a Th2 cell-type cytokine bias (αGalCer-C20:2 and -PGB1). The bar graph on the right shows relative potencies [(EC50 with presentation by CD1d.WT)/(EC50 with presentation by CD1d.TD)] of each glycolipid presented by CD1d.TD versus CD1d.WT.

Mentions: To visualize the kinetics and sites of mCD1d loading by αGalCer analogs in live cells, we analyzed intact JAWS II cells for surface staining with mAb L363 after 1, 4, or 16 hr of incubation with αGalCer-C26:0 or with either of two Th2 cell-type cytokine-biasing analogs (αGalCer-C20:2 or -C10:0) (Figure 4A). Strong staining of the plasma membrane was seen after 16 hr of incubation in all cases. However, only αGalCer-C20:2 or -C10:0 showed L363 staining at earlier time points, consistent with the rapid kinetics of loading observed by flow cytometry. To image earlier steps in glycolipid loading, we permeabilized and fixed cells prior to staining with L363 and with an antibody to the late endosomal and lysosomal marker LAMP-1 (Figure 4A). After incubation for 1 hr with glycolipids, L363 staining was observed at the plasma membrane for αGalCer-C20:2 and -C10:0, and no colocalization with LAMP-1 was present. Colocalization with LAMP-1 was observed for mCD1d complexes formed with these analogs only at the latest time point studied (16 hr), suggesting that they arrived at late endocytic compartments via recycling from the plasma membrane. In contrast, L363 staining of cells pulsed for 1 hr with αGalCer-26:0 was observed only in a punctate intracellular distribution that was completely distinct from LAMP-1 staining, indicating that the initial association with mCD1d occurred in an endocytic compartment separate from typical late endosomes and lysosomes. At 4 hr, these complexes began to colocalize with LAMP-1, and only at 16 hr could clear surface staining be seen along with the persistent intracellular staining in LAMP-1+ compartments (Figure 4A).


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)

Cell Surface versus Intracellular Loading of mCD1d with Different αGalCer Analogs(A) On the left, surface accumulation of CD1d-αGalCer complexes was studied with JAWS II cells harvested after incubation with the indicated glycolipids for 1, 4, or 16 hr and then stained without permeabilization with mAb L363 (red). Cells were then fixed, stained with DAPI for nuclei visualization (blue), and analyzed by confocal microscopy. On the right, the intracellular formation and trafficking of CD1d-αGalCer complexes were analyzed by treatment of JAWS II cells with αGalCer analogs for 1, 4, and 16 hr followed by fixation and permeabilization. The cells were stained for CD1d-αGalCer complexes (L363, red), late endosomes and lysosomes (anti-LAMP-1, green), and nuclei (DAPI, blue). Results are representative of three separate experiments. The scale bar represents 10 μm.(B) Murine iNKT cell hybridoma cells (DN3A4.1-2) were stimulated with αGalCer analogs. As APCs for these stimulations, we used A20 cells transfected with wild-type CD1d (CD1d.WT) or cytoplasmic-tail-deleted CD1d (CD1d.TD). Supernatant concentrations of IL-2 release were determined after 12 hr. IL-2 production over a range of concentrations is shown for αGalCer-C26:0 and two analogs that produce a Th2 cell-type cytokine bias (αGalCer-C20:2 and -PGB1). The bar graph on the right shows relative potencies [(EC50 with presentation by CD1d.WT)/(EC50 with presentation by CD1d.TD)] of each glycolipid presented by CD1d.TD versus CD1d.WT.
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fig4: Cell Surface versus Intracellular Loading of mCD1d with Different αGalCer Analogs(A) On the left, surface accumulation of CD1d-αGalCer complexes was studied with JAWS II cells harvested after incubation with the indicated glycolipids for 1, 4, or 16 hr and then stained without permeabilization with mAb L363 (red). Cells were then fixed, stained with DAPI for nuclei visualization (blue), and analyzed by confocal microscopy. On the right, the intracellular formation and trafficking of CD1d-αGalCer complexes were analyzed by treatment of JAWS II cells with αGalCer analogs for 1, 4, and 16 hr followed by fixation and permeabilization. The cells were stained for CD1d-αGalCer complexes (L363, red), late endosomes and lysosomes (anti-LAMP-1, green), and nuclei (DAPI, blue). Results are representative of three separate experiments. The scale bar represents 10 μm.(B) Murine iNKT cell hybridoma cells (DN3A4.1-2) were stimulated with αGalCer analogs. As APCs for these stimulations, we used A20 cells transfected with wild-type CD1d (CD1d.WT) or cytoplasmic-tail-deleted CD1d (CD1d.TD). Supernatant concentrations of IL-2 release were determined after 12 hr. IL-2 production over a range of concentrations is shown for αGalCer-C26:0 and two analogs that produce a Th2 cell-type cytokine bias (αGalCer-C20:2 and -PGB1). The bar graph on the right shows relative potencies [(EC50 with presentation by CD1d.WT)/(EC50 with presentation by CD1d.TD)] of each glycolipid presented by CD1d.TD versus CD1d.WT.
Mentions: To visualize the kinetics and sites of mCD1d loading by αGalCer analogs in live cells, we analyzed intact JAWS II cells for surface staining with mAb L363 after 1, 4, or 16 hr of incubation with αGalCer-C26:0 or with either of two Th2 cell-type cytokine-biasing analogs (αGalCer-C20:2 or -C10:0) (Figure 4A). Strong staining of the plasma membrane was seen after 16 hr of incubation in all cases. However, only αGalCer-C20:2 or -C10:0 showed L363 staining at earlier time points, consistent with the rapid kinetics of loading observed by flow cytometry. To image earlier steps in glycolipid loading, we permeabilized and fixed cells prior to staining with L363 and with an antibody to the late endosomal and lysosomal marker LAMP-1 (Figure 4A). After incubation for 1 hr with glycolipids, L363 staining was observed at the plasma membrane for αGalCer-C20:2 and -C10:0, and no colocalization with LAMP-1 was present. Colocalization with LAMP-1 was observed for mCD1d complexes formed with these analogs only at the latest time point studied (16 hr), suggesting that they arrived at late endocytic compartments via recycling from the plasma membrane. In contrast, L363 staining of cells pulsed for 1 hr with αGalCer-26:0 was observed only in a punctate intracellular distribution that was completely distinct from LAMP-1 staining, indicating that the initial association with mCD1d occurred in an endocytic compartment separate from typical late endosomes and lysosomes. At 4 hr, these complexes began to colocalize with LAMP-1, and only at 16 hr could clear surface staining be seen along with the persistent intracellular staining in LAMP-1+ compartments (Figure 4A).

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