Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of iNKT Cell TCR Interactions with αGalCer Analogs(A) Flow cytometry analysis of human and murine iNKT cells with αGalCer analog-CD1d tetramers. In the top row, murine iNKT cells from C57BL/6 splenocytes were costained with αGalCer analog-loaded or unloaded mCD1d tetramers conjugated with PE (x axis) and αGalCer-C26:0-loaded-mCD1d tetramers conjugated with APC (y axis). In the bottom row, human PBMCs were similarly costained with human glycolipid-loaded CD1d tetramers.(B) Binding avidity of glycolipid-mCD1d complexes to murine iNKT cell TCRs. Histograms at left show tetramer staining of NK1.1+ CD3+ T cells in C57BL/6 splenocytes with glycolipid-loaded tetramers at 20 nM. Mean fluorescence intensity (MFI) for staining of this population with tetramers at various concentrations is plotted (center), and the equilibrium dissociation constants (KD) were determined as the concentrations of glycolipid-mCD1d complexes required to yield 50% of maximal binding. Values are plotted as 1/KD, which is directly proportional to TCR avidity (right).(C) Similar experiments as in (B) for binding of αGalCer-hCD1d tetramers to a human iNKT cell clone (CD4+ clone HDD11). All results shown were representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2719696&req=5

fig2: Characterization of iNKT Cell TCR Interactions with αGalCer Analogs(A) Flow cytometry analysis of human and murine iNKT cells with αGalCer analog-CD1d tetramers. In the top row, murine iNKT cells from C57BL/6 splenocytes were costained with αGalCer analog-loaded or unloaded mCD1d tetramers conjugated with PE (x axis) and αGalCer-C26:0-loaded-mCD1d tetramers conjugated with APC (y axis). In the bottom row, human PBMCs were similarly costained with human glycolipid-loaded CD1d tetramers.(B) Binding avidity of glycolipid-mCD1d complexes to murine iNKT cell TCRs. Histograms at left show tetramer staining of NK1.1+ CD3+ T cells in C57BL/6 splenocytes with glycolipid-loaded tetramers at 20 nM. Mean fluorescence intensity (MFI) for staining of this population with tetramers at various concentrations is plotted (center), and the equilibrium dissociation constants (KD) were determined as the concentrations of glycolipid-mCD1d complexes required to yield 50% of maximal binding. Values are plotted as 1/KD, which is directly proportional to TCR avidity (right).(C) Similar experiments as in (B) for binding of αGalCer-hCD1d tetramers to a human iNKT cell clone (CD4+ clone HDD11). All results shown were representative of two independent experiments.

Mentions: The interactions of iNKT cell TCRs with CD1d molecules loaded with αGalCer analogs were analyzed with fluorescent murine and human CD1d tetramers. This demonstrated that iNKT cells recognizing each analog were also strongly reactive with αGalCer-C26:0, although staining patterns in some cases indicated differences in relative TCR avidities (Figure 2A). Equilibrium binding studies for measuring TCR avidities were carried out by incubation of human or murine iNKT cells with varying concentrations of αGalCer-analog-loaded tetramers and determining relative levels of cell-associated fluorescence (Stanic et al., 2003; Yu et al., 2005). A wide range of TCR avidities was observed for mouse CD1d (mCD1d) and human CD1d (hCD1d) complexes formed with different Th2 cell-type cytokine-biasing αGalCer analogs (Figures 2B and 2C). Notably, complexes loaded with some of these analogs showed avidities that were equal to or exceeded those of αGalCer-C26:0 or -C24:0 loaded complexes, arguing that low TCR avidity was not the main or sole determinant responsible for altered 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)

Characterization of iNKT Cell TCR Interactions with αGalCer Analogs(A) Flow cytometry analysis of human and murine iNKT cells with αGalCer analog-CD1d tetramers. In the top row, murine iNKT cells from C57BL/6 splenocytes were costained with αGalCer analog-loaded or unloaded mCD1d tetramers conjugated with PE (x axis) and αGalCer-C26:0-loaded-mCD1d tetramers conjugated with APC (y axis). In the bottom row, human PBMCs were similarly costained with human glycolipid-loaded CD1d tetramers.(B) Binding avidity of glycolipid-mCD1d complexes to murine iNKT cell TCRs. Histograms at left show tetramer staining of NK1.1+ CD3+ T cells in C57BL/6 splenocytes with glycolipid-loaded tetramers at 20 nM. Mean fluorescence intensity (MFI) for staining of this population with tetramers at various concentrations is plotted (center), and the equilibrium dissociation constants (KD) were determined as the concentrations of glycolipid-mCD1d complexes required to yield 50% of maximal binding. Values are plotted as 1/KD, which is directly proportional to TCR avidity (right).(C) Similar experiments as in (B) for binding of αGalCer-hCD1d tetramers to a human iNKT cell clone (CD4+ clone HDD11). All results shown were representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Characterization of iNKT Cell TCR Interactions with αGalCer Analogs(A) Flow cytometry analysis of human and murine iNKT cells with αGalCer analog-CD1d tetramers. In the top row, murine iNKT cells from C57BL/6 splenocytes were costained with αGalCer analog-loaded or unloaded mCD1d tetramers conjugated with PE (x axis) and αGalCer-C26:0-loaded-mCD1d tetramers conjugated with APC (y axis). In the bottom row, human PBMCs were similarly costained with human glycolipid-loaded CD1d tetramers.(B) Binding avidity of glycolipid-mCD1d complexes to murine iNKT cell TCRs. Histograms at left show tetramer staining of NK1.1+ CD3+ T cells in C57BL/6 splenocytes with glycolipid-loaded tetramers at 20 nM. Mean fluorescence intensity (MFI) for staining of this population with tetramers at various concentrations is plotted (center), and the equilibrium dissociation constants (KD) were determined as the concentrations of glycolipid-mCD1d complexes required to yield 50% of maximal binding. Values are plotted as 1/KD, which is directly proportional to TCR avidity (right).(C) Similar experiments as in (B) for binding of αGalCer-hCD1d tetramers to a human iNKT cell clone (CD4+ clone HDD11). All results shown were representative of two independent experiments.
Mentions: The interactions of iNKT cell TCRs with CD1d molecules loaded with αGalCer analogs were analyzed with fluorescent murine and human CD1d tetramers. This demonstrated that iNKT cells recognizing each analog were also strongly reactive with αGalCer-C26:0, although staining patterns in some cases indicated differences in relative TCR avidities (Figure 2A). Equilibrium binding studies for measuring TCR avidities were carried out by incubation of human or murine iNKT cells with varying concentrations of αGalCer-analog-loaded tetramers and determining relative levels of cell-associated fluorescence (Stanic et al., 2003; Yu et al., 2005). A wide range of TCR avidities was observed for mouse CD1d (mCD1d) and human CD1d (hCD1d) complexes formed with different Th2 cell-type cytokine-biasing αGalCer analogs (Figures 2B and 2C). Notably, complexes loaded with some of these analogs showed avidities that were equal to or exceeded those of αGalCer-C26:0 or -C24:0 loaded complexes, arguing that low TCR avidity was not the main or sole determinant responsible for altered 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