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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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Direct Analysis of Presentation of αGalCer Analogs with Complex-Specific mAb Staining(A) Mouse splenocytes cultured with either vehicle only (0.02% DMSO) or with the indicated αGalCer analogs (100 nM) were harvested after 1 hr or 19 hr of incubation and stained with mAb L363 or anti-CD1d mAb 1B1 and with mAbs specific for CD11c, CD19, and F4/80 (Figure S4). Filled histograms show L363 staining of cells cultured with vehicle only, and open histograms show cells cultured with indicated glycolipids. At the left, the total CD1d staining with mAb 1B1 (open histograms) compared to isotype control mAb (filled histograms) are shown on cells cultured for 19 hr with DMSO only.(B) JAWS II cells were incubated with glycolipids (250 nM) for various times ranging from 0.5–18 hr as indicated, stained with mAb L363, and analyzed by flow cytometry. Dashed line histograms show L363 staining of cells incubated with medium only, and bold histograms show L363 staining of cells incubated with αGalCer analogs.(C) JAWS II cells were pulsed with indicated αGalCer analogs (500 nM) for 1.5 hr, washed extensively, and recultured at 37°C for various chase times. Upon harvesting, cells were placed on ice and subsequently stained with mAb L363 for flow cytometry analysis. Histogram overlays show L363 staining of cells pulsed initially with vehicle only (DMSO, dashed histograms) or with αGalCer analog (bold histograms). The graph on the right shows MFI of L363 staining at each chase time represented as percent of the maximum value measured during the course of the experiment. All results shown (A, B, and C) were representative of at least two independent experiments.
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fig3: Direct Analysis of Presentation of αGalCer Analogs with Complex-Specific mAb Staining(A) Mouse splenocytes cultured with either vehicle only (0.02% DMSO) or with the indicated αGalCer analogs (100 nM) were harvested after 1 hr or 19 hr of incubation and stained with mAb L363 or anti-CD1d mAb 1B1 and with mAbs specific for CD11c, CD19, and F4/80 (Figure S4). Filled histograms show L363 staining of cells cultured with vehicle only, and open histograms show cells cultured with indicated glycolipids. At the left, the total CD1d staining with mAb 1B1 (open histograms) compared to isotype control mAb (filled histograms) are shown on cells cultured for 19 hr with DMSO only.(B) JAWS II cells were incubated with glycolipids (250 nM) for various times ranging from 0.5–18 hr as indicated, stained with mAb L363, and analyzed by flow cytometry. Dashed line histograms show L363 staining of cells incubated with medium only, and bold histograms show L363 staining of cells incubated with αGalCer analogs.(C) JAWS II cells were pulsed with indicated αGalCer analogs (500 nM) for 1.5 hr, washed extensively, and recultured at 37°C for various chase times. Upon harvesting, cells were placed on ice and subsequently stained with mAb L363 for flow cytometry analysis. Histogram overlays show L363 staining of cells pulsed initially with vehicle only (DMSO, dashed histograms) or with αGalCer analog (bold histograms). The graph on the right shows MFI of L363 staining at each chase time represented as percent of the maximum value measured during the course of the experiment. All results shown (A, B, and C) were representative of at least two independent experiments.

Mentions: A previous study suggested that an iNKT cell response biased toward production of Th2 cell-type cytokines could result from preferential presentation of αGalCer by B lymphocytes (Bezbradica et al., 2005). We studied this possibility directly by using a monoclonal antibody (L363) that specifically recognizes complexes of αGalCer bound to mCD1d (Yu et al., 2007). Mouse splenocytes were cultured with either αGalCer-C26:0 or representative Th2 cell-type cytokine-biasing analogs, and L363 staining was assessed on various cell types (Figure 3A). This revealed that all αGalCer analogs were presented most prominently on CD11c+ DCs, and only one of the two analogs that induced a Th2 cell-type cytokine response showed clearly detectable presentation by B cells even after prolonged incubation (19 hr). However, the extremely early appearance of L363 staining on cells exposed to αGalCer-C10:0 suggested that rapid kinetics of presentation might be a key feature of αGalCer analogs that induce a bias toward Th2 cell-type cytokines. This was investigated further with more detailed kinetic studies in which immortalized mouse DC line JAWS II was cultured with each glycolipid and assessed for L363 surface staining at 0.5, 4, and 18 hr (Figure 3B). There was a striking correlation between rapid kinetics of glycolipid association with mCD1d and the ability to induce a Th2 cell-type cytokine response. For analogs that induced a mixed cytokine response (αGalCer-C26:0 and -C24:0), strong L363 staining was detected only at 18 hr. In contrast, most analogs inducing Th2 cell-type cytokine responses gave strong L363 staining by 0.5 hr, which increased modestly or not at all with longer incubation. The single exception was OCH, which in general gave very weak L363 reactivity, although this was also apparent by 4 hr. A pulse-chase analysis revealed that mCD1d-αGalCer surface complexes formed slowly with αGalCer-C26:0 and continued to accumulate throughout the 8 hr chase (Figure 3C), whereas with αGalCer-C20:2 or -C10:0, the complexes formed rapidly and began declining immediately during the chase period. Experiments using responses of iNKT cells to analyze the kinetics of cell-surface appearance gave analogous results (Figure S5). Thus, although our findings did not provide evidence for presentation by different APC types as a major factor accounting for the altered cytokine responses to different analogs of αGalCer, they revealed a consistent tendency for glycolipids that induced Th2 cell-type biased cytokine responses to be presented with more rapid kinetics compared to αGalCer analogs that induced mixed Th1 and Th2 cell-type cytokine responses.


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)

Direct Analysis of Presentation of αGalCer Analogs with Complex-Specific mAb Staining(A) Mouse splenocytes cultured with either vehicle only (0.02% DMSO) or with the indicated αGalCer analogs (100 nM) were harvested after 1 hr or 19 hr of incubation and stained with mAb L363 or anti-CD1d mAb 1B1 and with mAbs specific for CD11c, CD19, and F4/80 (Figure S4). Filled histograms show L363 staining of cells cultured with vehicle only, and open histograms show cells cultured with indicated glycolipids. At the left, the total CD1d staining with mAb 1B1 (open histograms) compared to isotype control mAb (filled histograms) are shown on cells cultured for 19 hr with DMSO only.(B) JAWS II cells were incubated with glycolipids (250 nM) for various times ranging from 0.5–18 hr as indicated, stained with mAb L363, and analyzed by flow cytometry. Dashed line histograms show L363 staining of cells incubated with medium only, and bold histograms show L363 staining of cells incubated with αGalCer analogs.(C) JAWS II cells were pulsed with indicated αGalCer analogs (500 nM) for 1.5 hr, washed extensively, and recultured at 37°C for various chase times. Upon harvesting, cells were placed on ice and subsequently stained with mAb L363 for flow cytometry analysis. Histogram overlays show L363 staining of cells pulsed initially with vehicle only (DMSO, dashed histograms) or with αGalCer analog (bold histograms). The graph on the right shows MFI of L363 staining at each chase time represented as percent of the maximum value measured during the course of the experiment. All results shown (A, B, and C) were representative of at least two independent experiments.
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Related In: Results  -  Collection

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fig3: Direct Analysis of Presentation of αGalCer Analogs with Complex-Specific mAb Staining(A) Mouse splenocytes cultured with either vehicle only (0.02% DMSO) or with the indicated αGalCer analogs (100 nM) were harvested after 1 hr or 19 hr of incubation and stained with mAb L363 or anti-CD1d mAb 1B1 and with mAbs specific for CD11c, CD19, and F4/80 (Figure S4). Filled histograms show L363 staining of cells cultured with vehicle only, and open histograms show cells cultured with indicated glycolipids. At the left, the total CD1d staining with mAb 1B1 (open histograms) compared to isotype control mAb (filled histograms) are shown on cells cultured for 19 hr with DMSO only.(B) JAWS II cells were incubated with glycolipids (250 nM) for various times ranging from 0.5–18 hr as indicated, stained with mAb L363, and analyzed by flow cytometry. Dashed line histograms show L363 staining of cells incubated with medium only, and bold histograms show L363 staining of cells incubated with αGalCer analogs.(C) JAWS II cells were pulsed with indicated αGalCer analogs (500 nM) for 1.5 hr, washed extensively, and recultured at 37°C for various chase times. Upon harvesting, cells were placed on ice and subsequently stained with mAb L363 for flow cytometry analysis. Histogram overlays show L363 staining of cells pulsed initially with vehicle only (DMSO, dashed histograms) or with αGalCer analog (bold histograms). The graph on the right shows MFI of L363 staining at each chase time represented as percent of the maximum value measured during the course of the experiment. All results shown (A, B, and C) were representative of at least two independent experiments.
Mentions: A previous study suggested that an iNKT cell response biased toward production of Th2 cell-type cytokines could result from preferential presentation of αGalCer by B lymphocytes (Bezbradica et al., 2005). We studied this possibility directly by using a monoclonal antibody (L363) that specifically recognizes complexes of αGalCer bound to mCD1d (Yu et al., 2007). Mouse splenocytes were cultured with either αGalCer-C26:0 or representative Th2 cell-type cytokine-biasing analogs, and L363 staining was assessed on various cell types (Figure 3A). This revealed that all αGalCer analogs were presented most prominently on CD11c+ DCs, and only one of the two analogs that induced a Th2 cell-type cytokine response showed clearly detectable presentation by B cells even after prolonged incubation (19 hr). However, the extremely early appearance of L363 staining on cells exposed to αGalCer-C10:0 suggested that rapid kinetics of presentation might be a key feature of αGalCer analogs that induce a bias toward Th2 cell-type cytokines. This was investigated further with more detailed kinetic studies in which immortalized mouse DC line JAWS II was cultured with each glycolipid and assessed for L363 surface staining at 0.5, 4, and 18 hr (Figure 3B). There was a striking correlation between rapid kinetics of glycolipid association with mCD1d and the ability to induce a Th2 cell-type cytokine response. For analogs that induced a mixed cytokine response (αGalCer-C26:0 and -C24:0), strong L363 staining was detected only at 18 hr. In contrast, most analogs inducing Th2 cell-type cytokine responses gave strong L363 staining by 0.5 hr, which increased modestly or not at all with longer incubation. The single exception was OCH, which in general gave very weak L363 reactivity, although this was also apparent by 4 hr. A pulse-chase analysis revealed that mCD1d-αGalCer surface complexes formed slowly with αGalCer-C26:0 and continued to accumulate throughout the 8 hr chase (Figure 3C), whereas with αGalCer-C20:2 or -C10:0, the complexes formed rapidly and began declining immediately during the chase period. Experiments using responses of iNKT cells to analyze the kinetics of cell-surface appearance gave analogous results (Figure S5). Thus, although our findings did not provide evidence for presentation by different APC types as a major factor accounting for the altered cytokine responses to different analogs of αGalCer, they revealed a consistent tendency for glycolipids that induced Th2 cell-type biased cytokine responses to be presented with more rapid kinetics compared to αGalCer analogs that induced mixed Th1 and Th2 cell-type cytokine responses.

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