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Galectin-9 controls CD40 signaling through a Tim-3 independent mechanism and redirects the cytokine profile of pathogenic T cells in autoimmunity.

Vaitaitis GM, Wagner DH - PLoS ONE (2012)

Bottom Line: Galectins interact with carbohydrates on proteins to effect such signaling alterations.Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells.Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4(lo)CD40(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Webb-Waring Center, University of Colorado Denver, Aurora, Colorado, United States of America.

ABSTRACT
While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4(lo)CD40(+) effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4(lo)CD40(+) T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones.

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Galectin-9 exerts its proliferation inhibitory and cell death inducing effects on CD4loCD40+ T cells via the carbohydrate recognition domains of the protein.CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were CFSE labeled. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of 7.5 µg/ml galectin-9 (gal-9) for 4 days. Lactose was added as a competitor for the CRD of galectin-9. (A) Cell cluster formation was observed. (B) Proliferation was measured by CFSE dilution. (C) Bar graphs representing proliferation and necrotic cell death. Asterisks in C denote significant differences determined by one-way Anova; ns – not significant; ** – P between 0.001 and 0.01; *** – P <0.001; **** – P<0.0001. Data represent experiments done on three individual mice of different ages.
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pone-0038708-g002: Galectin-9 exerts its proliferation inhibitory and cell death inducing effects on CD4loCD40+ T cells via the carbohydrate recognition domains of the protein.CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were CFSE labeled. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of 7.5 µg/ml galectin-9 (gal-9) for 4 days. Lactose was added as a competitor for the CRD of galectin-9. (A) Cell cluster formation was observed. (B) Proliferation was measured by CFSE dilution. (C) Bar graphs representing proliferation and necrotic cell death. Asterisks in C denote significant differences determined by one-way Anova; ns – not significant; ** – P between 0.001 and 0.01; *** – P <0.001; **** – P<0.0001. Data represent experiments done on three individual mice of different ages.

Mentions: Galectin-9 consists of two different carbohydrate recognition domains (CRD) connected to each other with a linker polypeptide chain [24]. The interaction of galectin-9 with its receptors is dependent on the CRD and carbohydrates on the receptor proteins. Therefore we determined whether the galectin-9 effects on CD4loCD40+ T cells were preventable by lactose, a competitive substrate that inhibits the interaction of galectins with their receptors. We have demonstrated previously that NOD CD4loCD40+ T cells form distinct ball shaped clusters when CD40 engaged [17]. When galectin-9 was added to CD40 engaged NOD CD4loCD40+ T cells those clusters no longer formed (Fig. 2A). However, when lactose was added the cluster formation was regained. Similarly, when proliferation was assessed, the galectin-9 inhibition of CD40 induced proliferation was prevented by the addition of lactose (Fig. 2B, bottom histogram and 2C). Interestingly, the higher concentration, 30 mM, of lactose inhibited the CD40 induced proliferation (Fig. 2B, middle histogram and 2C). This could be due to that other galectins may be involved in survival and proliferative responses and would be competed for by lactose as well. When cell death was assessed, 30 mM lactose, but not 10 mM, induced increased necrotic cell death in the isotype treated and CD40 engaged cells (Fig. 2C) again demonstrating that other galectins may be involved in survival. When lactose was added to cells treated with CD40 engagement plus galectin-9, the galectin-9 induced cell death was prevented demonstrating that the CRD of galectin-9 is involved in the mechanism leading to cell death (Fig. 2C).


Galectin-9 controls CD40 signaling through a Tim-3 independent mechanism and redirects the cytokine profile of pathogenic T cells in autoimmunity.

Vaitaitis GM, Wagner DH - PLoS ONE (2012)

Galectin-9 exerts its proliferation inhibitory and cell death inducing effects on CD4loCD40+ T cells via the carbohydrate recognition domains of the protein.CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were CFSE labeled. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of 7.5 µg/ml galectin-9 (gal-9) for 4 days. Lactose was added as a competitor for the CRD of galectin-9. (A) Cell cluster formation was observed. (B) Proliferation was measured by CFSE dilution. (C) Bar graphs representing proliferation and necrotic cell death. Asterisks in C denote significant differences determined by one-way Anova; ns – not significant; ** – P between 0.001 and 0.01; *** – P <0.001; **** – P<0.0001. Data represent experiments done on three individual mice of different ages.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369903&req=5

pone-0038708-g002: Galectin-9 exerts its proliferation inhibitory and cell death inducing effects on CD4loCD40+ T cells via the carbohydrate recognition domains of the protein.CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were CFSE labeled. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of 7.5 µg/ml galectin-9 (gal-9) for 4 days. Lactose was added as a competitor for the CRD of galectin-9. (A) Cell cluster formation was observed. (B) Proliferation was measured by CFSE dilution. (C) Bar graphs representing proliferation and necrotic cell death. Asterisks in C denote significant differences determined by one-way Anova; ns – not significant; ** – P between 0.001 and 0.01; *** – P <0.001; **** – P<0.0001. Data represent experiments done on three individual mice of different ages.
Mentions: Galectin-9 consists of two different carbohydrate recognition domains (CRD) connected to each other with a linker polypeptide chain [24]. The interaction of galectin-9 with its receptors is dependent on the CRD and carbohydrates on the receptor proteins. Therefore we determined whether the galectin-9 effects on CD4loCD40+ T cells were preventable by lactose, a competitive substrate that inhibits the interaction of galectins with their receptors. We have demonstrated previously that NOD CD4loCD40+ T cells form distinct ball shaped clusters when CD40 engaged [17]. When galectin-9 was added to CD40 engaged NOD CD4loCD40+ T cells those clusters no longer formed (Fig. 2A). However, when lactose was added the cluster formation was regained. Similarly, when proliferation was assessed, the galectin-9 inhibition of CD40 induced proliferation was prevented by the addition of lactose (Fig. 2B, bottom histogram and 2C). Interestingly, the higher concentration, 30 mM, of lactose inhibited the CD40 induced proliferation (Fig. 2B, middle histogram and 2C). This could be due to that other galectins may be involved in survival and proliferative responses and would be competed for by lactose as well. When cell death was assessed, 30 mM lactose, but not 10 mM, induced increased necrotic cell death in the isotype treated and CD40 engaged cells (Fig. 2C) again demonstrating that other galectins may be involved in survival. When lactose was added to cells treated with CD40 engagement plus galectin-9, the galectin-9 induced cell death was prevented demonstrating that the CRD of galectin-9 is involved in the mechanism leading to cell death (Fig. 2C).

Bottom Line: Galectins interact with carbohydrates on proteins to effect such signaling alterations.Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells.Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4(lo)CD40(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Webb-Waring Center, University of Colorado Denver, Aurora, Colorado, United States of America.

ABSTRACT
While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4(lo)CD40(+) effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4(lo)CD40(+) T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones.

Show MeSH
Related in: MedlinePlus