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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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Requirement of PKCθ for CAG-mediated proliferation. (A-C) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were stimulated with anti-CD3/CD28–coated beads, and after removal of beads they were cultured again in the presence or absence of galectin-4 for 1 (A) and 3 h (B). They were pulsed with BrdU 1 h before the analysis and subjected to flow cytometric analysis. The percentage of BrdU+ cells is summarized in C. *, P < 0.01 (n = 4–7). (D) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were pretreated with KT5720 (left) or rotterin (right), and they were then stimulated with anti-CD3/CD28–coated beads. After removal of beads, they were cultured in the presence of galectin-4 for 3 h, with 1-h pulse with BrdU, and subjected to flow cytometric analysis for evaluation of BrdU incorporation (top) and galectin-4 binding (bottom). Data shown are one representation of two independent experiments. (E and F) Purified CD4+CD45RBlow memory T cells from the spleen of GFP Tg versus PKCθ−/− mice were co-transferred into βIL-2 DKO mice. The intensity of galectin-4 binding on (E) and the proliferative response of (F) reconstituted GFP+ CD4+ (PKCθ-intact) versus GFP− CD4+ (PKCθ-deficient) cells in the same recipient colon are shown. The mean (n = 4) of percentages of BrdU+ cells in GFP+ (PKCθ-intact) versus GFP− (PKCθ-deficient) CD4+ T cells are summarized in G. *, P < 0.01.
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fig8: Requirement of PKCθ for CAG-mediated proliferation. (A-C) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were stimulated with anti-CD3/CD28–coated beads, and after removal of beads they were cultured again in the presence or absence of galectin-4 for 1 (A) and 3 h (B). They were pulsed with BrdU 1 h before the analysis and subjected to flow cytometric analysis. The percentage of BrdU+ cells is summarized in C. *, P < 0.01 (n = 4–7). (D) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were pretreated with KT5720 (left) or rotterin (right), and they were then stimulated with anti-CD3/CD28–coated beads. After removal of beads, they were cultured in the presence of galectin-4 for 3 h, with 1-h pulse with BrdU, and subjected to flow cytometric analysis for evaluation of BrdU incorporation (top) and galectin-4 binding (bottom). Data shown are one representation of two independent experiments. (E and F) Purified CD4+CD45RBlow memory T cells from the spleen of GFP Tg versus PKCθ−/− mice were co-transferred into βIL-2 DKO mice. The intensity of galectin-4 binding on (E) and the proliferative response of (F) reconstituted GFP+ CD4+ (PKCθ-intact) versus GFP− CD4+ (PKCθ-deficient) cells in the same recipient colon are shown. The mean (n = 4) of percentages of BrdU+ cells in GFP+ (PKCθ-intact) versus GFP− (PKCθ-deficient) CD4+ T cells are summarized in G. *, P < 0.01.

Mentions: In a final set of experiments, we examined whether the sustained PKCθ activation in colonic CD4+ T cells by CAG development promotes the proliferative response. To do so, colonic memory CD4+ T cells were stimulated as shown in Fig. 7 D, and the activated cells were cultured again in the presence of galectin-4 for 1 or 3 h. At 1 h before analysis, cells were pulsed with BrdU. As shown in Fig. 8 (A and C), similar levels of proliferation were observed in WT- versus C2GnT tg–derived CD4+ T cells at 1 h after stimulation. However, at 3 h after stimulation, much less proliferation was seen in C2GnT tg–derived CD4+ T cells compared with WT-derived CD4+ T cells (Fig. 8, B and C). In addition, the proliferative response of WT-derived CD4+ T cells was reduced when galectin-4 was omitted from the culture (Fig. 8, B and C).


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)

Requirement of PKCθ for CAG-mediated proliferation. (A-C) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were stimulated with anti-CD3/CD28–coated beads, and after removal of beads they were cultured again in the presence or absence of galectin-4 for 1 (A) and 3 h (B). They were pulsed with BrdU 1 h before the analysis and subjected to flow cytometric analysis. The percentage of BrdU+ cells is summarized in C. *, P < 0.01 (n = 4–7). (D) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were pretreated with KT5720 (left) or rotterin (right), and they were then stimulated with anti-CD3/CD28–coated beads. After removal of beads, they were cultured in the presence of galectin-4 for 3 h, with 1-h pulse with BrdU, and subjected to flow cytometric analysis for evaluation of BrdU incorporation (top) and galectin-4 binding (bottom). Data shown are one representation of two independent experiments. (E and F) Purified CD4+CD45RBlow memory T cells from the spleen of GFP Tg versus PKCθ−/− mice were co-transferred into βIL-2 DKO mice. The intensity of galectin-4 binding on (E) and the proliferative response of (F) reconstituted GFP+ CD4+ (PKCθ-intact) versus GFP− CD4+ (PKCθ-deficient) cells in the same recipient colon are shown. The mean (n = 4) of percentages of BrdU+ cells in GFP+ (PKCθ-intact) versus GFP− (PKCθ-deficient) CD4+ T cells are summarized in G. *, P < 0.01.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: Requirement of PKCθ for CAG-mediated proliferation. (A-C) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were stimulated with anti-CD3/CD28–coated beads, and after removal of beads they were cultured again in the presence or absence of galectin-4 for 1 (A) and 3 h (B). They were pulsed with BrdU 1 h before the analysis and subjected to flow cytometric analysis. The percentage of BrdU+ cells is summarized in C. *, P < 0.01 (n = 4–7). (D) Purified colonic CD4+ T cells from βIL-2 DKO mice with reconstitution of WT-derived (WT>βIL2) versus T/C2GnT tg–derived (C2GnT>βIL2) memory CD4+ T cells were pretreated with KT5720 (left) or rotterin (right), and they were then stimulated with anti-CD3/CD28–coated beads. After removal of beads, they were cultured in the presence of galectin-4 for 3 h, with 1-h pulse with BrdU, and subjected to flow cytometric analysis for evaluation of BrdU incorporation (top) and galectin-4 binding (bottom). Data shown are one representation of two independent experiments. (E and F) Purified CD4+CD45RBlow memory T cells from the spleen of GFP Tg versus PKCθ−/− mice were co-transferred into βIL-2 DKO mice. The intensity of galectin-4 binding on (E) and the proliferative response of (F) reconstituted GFP+ CD4+ (PKCθ-intact) versus GFP− CD4+ (PKCθ-deficient) cells in the same recipient colon are shown. The mean (n = 4) of percentages of BrdU+ cells in GFP+ (PKCθ-intact) versus GFP− (PKCθ-deficient) CD4+ T cells are summarized in G. *, P < 0.01.
Mentions: In a final set of experiments, we examined whether the sustained PKCθ activation in colonic CD4+ T cells by CAG development promotes the proliferative response. To do so, colonic memory CD4+ T cells were stimulated as shown in Fig. 7 D, and the activated cells were cultured again in the presence of galectin-4 for 1 or 3 h. At 1 h before analysis, cells were pulsed with BrdU. As shown in Fig. 8 (A and C), similar levels of proliferation were observed in WT- versus C2GnT tg–derived CD4+ T cells at 1 h after stimulation. However, at 3 h after stimulation, much less proliferation was seen in C2GnT tg–derived CD4+ T cells compared with WT-derived CD4+ T cells (Fig. 8, B and C). In addition, the proliferative response of WT-derived CD4+ T cells was reduced when galectin-4 was omitted from the culture (Fig. 8, B and C).

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