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Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells.

Nishida A, Nagahama K, Imaeda H, Ogawa A, Lau CW, Kobayashi T, Hisamatsu T, Preffer FI, Mizoguchi E, Ikeuchi H, Hibi T, Fukuda M, Andoh A, Blumberg RS, Mizoguchi A - J. Exp. Med. (2012)

Bottom Line: The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan.Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations.Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Boston, MA 02114, USA.

ABSTRACT
Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

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Contribution of CAG for stabilization of PKCθ activity, (A) Purified CD4+ T cells from the inflamed colon were stained with anti-PKCθ (green, left) and ant-Fam62a (red, middle) antibodies. The merge image is shown on the right. Data shown are one representation of three 3 independent experiments. Bars, 15 µm. (B) Memory CD4+ T cells from GFP tg mice and T/C2GnT tg mice were co-transferred into βIL-2 DKO mice, and the intensity of CT-binding on GFP+ (line) versus GFP− (solid) CD4+ T cells from the same recipient colon was examined. Data shown are one representation of three independent experiments. (C–E) CD4+ T cells (Fresh in C) were purified from the colon of recipient βIL-2 DKO mice with transfer of WT-derived versus C2GnT Tg (C2GnT)–derived memory CD4+ T cells. Purified CD4+ T cells were stimulated with ant-CD3/CD28 coated beads, and after removal of beads they were cultured again in the presence of galectin-4 for 1 (1 h in D) and 3 h (3 h in D). Lipid rafts were precipitated using CT-coupled beads and subjected to detection of PKCθ (top). The bottom panels show the Coomassie blue staining of total protein subjected to CT-precipitation. The CD4+ T cells cultured for 3 h were stained with anti-PKCθ antibodies and subjected to confocal microscopic analysis (E). Bars, 15 µm. Data shown are one representation of three independent experiments.
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fig7: Contribution of CAG for stabilization of PKCθ activity, (A) Purified CD4+ T cells from the inflamed colon were stained with anti-PKCθ (green, left) and ant-Fam62a (red, middle) antibodies. The merge image is shown on the right. Data shown are one representation of three 3 independent experiments. Bars, 15 µm. (B) Memory CD4+ T cells from GFP tg mice and T/C2GnT tg mice were co-transferred into βIL-2 DKO mice, and the intensity of CT-binding on GFP+ (line) versus GFP− (solid) CD4+ T cells from the same recipient colon was examined. Data shown are one representation of three independent experiments. (C–E) CD4+ T cells (Fresh in C) were purified from the colon of recipient βIL-2 DKO mice with transfer of WT-derived versus C2GnT Tg (C2GnT)–derived memory CD4+ T cells. Purified CD4+ T cells were stimulated with ant-CD3/CD28 coated beads, and after removal of beads they were cultured again in the presence of galectin-4 for 1 (1 h in D) and 3 h (3 h in D). Lipid rafts were precipitated using CT-coupled beads and subjected to detection of PKCθ (top). The bottom panels show the Coomassie blue staining of total protein subjected to CT-precipitation. The CD4+ T cells cultured for 3 h were stained with anti-PKCθ antibodies and subjected to confocal microscopic analysis (E). Bars, 15 µm. Data shown are one representation of three independent experiments.

Mentions: Galectin-4 has previously been demonstrated to stabilize lipid rafts on epithelial cells for generation of super rafts (Braccia et al., 2003). The immunological synapse (IS), which is built on the lipid raft characterized by detergent insolubility, serves as a key component providing signals for T cell stimulation and proliferation (Xavier et al., 1998; Arendt et al., 2002). We therefore biochemically determined whether galectin-4 binds to lipid rafts associated with the IS. In brief, purified CD4+ T cells were obtained from the inflamed colon, lysed as previously described (Xavier et al., 1998), and precipitated with galectin-4–coupled beads. The precipitants were then subjected to analysis with a liquid chromatography tandem mass spectrometry. The protein complex precipitated by galectin-4 included formin-like 1, vinculin, actin-polymerization Arpc3, and Rab11, all of which have been identified to localize within the IS (Gomez et al., 2007; Nolz et al., 2007). Interestingly, galectin-4 also precipitated Fam62a (five-C2 domain-containing protein); we confirmed Fam62a has accumulated within the IS by confocal microscopy (Fig. 7 A). These findings indicate that galectin-4 interacts with CAG in the detergent-insoluble region of the cell membrane (lipid raft) that is in association with the IS.


Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells.

Nishida A, Nagahama K, Imaeda H, Ogawa A, Lau CW, Kobayashi T, Hisamatsu T, Preffer FI, Mizoguchi E, Ikeuchi H, Hibi T, Fukuda M, Andoh A, Blumberg RS, Mizoguchi A - J. Exp. Med. (2012)

Contribution of CAG for stabilization of PKCθ activity, (A) Purified CD4+ T cells from the inflamed colon were stained with anti-PKCθ (green, left) and ant-Fam62a (red, middle) antibodies. The merge image is shown on the right. Data shown are one representation of three 3 independent experiments. Bars, 15 µm. (B) Memory CD4+ T cells from GFP tg mice and T/C2GnT tg mice were co-transferred into βIL-2 DKO mice, and the intensity of CT-binding on GFP+ (line) versus GFP− (solid) CD4+ T cells from the same recipient colon was examined. Data shown are one representation of three independent experiments. (C–E) CD4+ T cells (Fresh in C) were purified from the colon of recipient βIL-2 DKO mice with transfer of WT-derived versus C2GnT Tg (C2GnT)–derived memory CD4+ T cells. Purified CD4+ T cells were stimulated with ant-CD3/CD28 coated beads, and after removal of beads they were cultured again in the presence of galectin-4 for 1 (1 h in D) and 3 h (3 h in D). Lipid rafts were precipitated using CT-coupled beads and subjected to detection of PKCθ (top). The bottom panels show the Coomassie blue staining of total protein subjected to CT-precipitation. The CD4+ T cells cultured for 3 h were stained with anti-PKCθ antibodies and subjected to confocal microscopic analysis (E). Bars, 15 µm. Data shown are one representation of three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526363&req=5

fig7: Contribution of CAG for stabilization of PKCθ activity, (A) Purified CD4+ T cells from the inflamed colon were stained with anti-PKCθ (green, left) and ant-Fam62a (red, middle) antibodies. The merge image is shown on the right. Data shown are one representation of three 3 independent experiments. Bars, 15 µm. (B) Memory CD4+ T cells from GFP tg mice and T/C2GnT tg mice were co-transferred into βIL-2 DKO mice, and the intensity of CT-binding on GFP+ (line) versus GFP− (solid) CD4+ T cells from the same recipient colon was examined. Data shown are one representation of three independent experiments. (C–E) CD4+ T cells (Fresh in C) were purified from the colon of recipient βIL-2 DKO mice with transfer of WT-derived versus C2GnT Tg (C2GnT)–derived memory CD4+ T cells. Purified CD4+ T cells were stimulated with ant-CD3/CD28 coated beads, and after removal of beads they were cultured again in the presence of galectin-4 for 1 (1 h in D) and 3 h (3 h in D). Lipid rafts were precipitated using CT-coupled beads and subjected to detection of PKCθ (top). The bottom panels show the Coomassie blue staining of total protein subjected to CT-precipitation. The CD4+ T cells cultured for 3 h were stained with anti-PKCθ antibodies and subjected to confocal microscopic analysis (E). Bars, 15 µm. Data shown are one representation of three independent experiments.
Mentions: Galectin-4 has previously been demonstrated to stabilize lipid rafts on epithelial cells for generation of super rafts (Braccia et al., 2003). The immunological synapse (IS), which is built on the lipid raft characterized by detergent insolubility, serves as a key component providing signals for T cell stimulation and proliferation (Xavier et al., 1998; Arendt et al., 2002). We therefore biochemically determined whether galectin-4 binds to lipid rafts associated with the IS. In brief, purified CD4+ T cells were obtained from the inflamed colon, lysed as previously described (Xavier et al., 1998), and precipitated with galectin-4–coupled beads. The precipitants were then subjected to analysis with a liquid chromatography tandem mass spectrometry. The protein complex precipitated by galectin-4 included formin-like 1, vinculin, actin-polymerization Arpc3, and Rab11, all of which have been identified to localize within the IS (Gomez et al., 2007; Nolz et al., 2007). Interestingly, galectin-4 also precipitated Fam62a (five-C2 domain-containing protein); we confirmed Fam62a has accumulated within the IS by confocal microscopy (Fig. 7 A). These findings indicate that galectin-4 interacts with CAG in the detergent-insoluble region of the cell membrane (lipid raft) that is in association with the IS.

Bottom Line: The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan.Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations.Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Boston, MA 02114, USA.

ABSTRACT
Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

Show MeSH
Related in: MedlinePlus