Limits...
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.

Show MeSH

Related in: MedlinePlus

Galectin-9 causes autoimmune CD4loCD40+ T cells to appear more like CD4hi T cells and increases CD3 induced proliferation.CD4loCD40+ and CD4hi T cells were sorted from 7–13 weeks old female NOD spleens. (A) CD4loCD40+ T cells were isotype treated (Isotype) or CD40-stimulated for 2 days in the absence/presence of indicated concentrations of galectin-9 then photographed under a microscope. (B) NOD CD4loCD40+ T cells were treated for 2 hours with 2.5 µg/ml galectin-9 then stained for CD3, CD4, TCR, CD5 and CD8. Tinted – untreated; black line – 2.5 µg/ml galectin-9. Gates were set based on appropriate isotype controls. Below each histogram is a corresponding bar graph depicting the cumulative data. (C) NOD CD4hi T cells were either stained immediately for CD28 or CD40 (left two histograms; grey tinted – stain-isotype; black line – CD28 or CD40 as indicated in figure; percentages are means with SEM) or isotype treated or stimulated with CD3+CD28+CD40 overnight then stained for CD4 (bar graph and right histogram; grey tinted – stain-isotype; black line – isotype treated; dotted line – CD3+CD28+CD40 stimulated). (D) NOD CD4loCD40+ T cells were CFSE labeled then isotype treated (Isotype) or CD3-stimulated (CD3XL) in the absence/presence of indicated concentrations of galectin-9 (gal-9) for 8 days. Proliferation was measured by CFSE dilution. All bar graphs in this figure depict means with SEM. Asterisks denote significant differences determined by one- or two-way Anova as appropriate; ns – not significant; * – P between 0.01 and 0.05; ** – P <0.01; *** – P <0.001; **** – P<0.0001. Experiments were done at least three separate times.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369903&req=5

pone-0038708-g005: Galectin-9 causes autoimmune CD4loCD40+ T cells to appear more like CD4hi T cells and increases CD3 induced proliferation.CD4loCD40+ and CD4hi T cells were sorted from 7–13 weeks old female NOD spleens. (A) CD4loCD40+ T cells were isotype treated (Isotype) or CD40-stimulated for 2 days in the absence/presence of indicated concentrations of galectin-9 then photographed under a microscope. (B) NOD CD4loCD40+ T cells were treated for 2 hours with 2.5 µg/ml galectin-9 then stained for CD3, CD4, TCR, CD5 and CD8. Tinted – untreated; black line – 2.5 µg/ml galectin-9. Gates were set based on appropriate isotype controls. Below each histogram is a corresponding bar graph depicting the cumulative data. (C) NOD CD4hi T cells were either stained immediately for CD28 or CD40 (left two histograms; grey tinted – stain-isotype; black line – CD28 or CD40 as indicated in figure; percentages are means with SEM) or isotype treated or stimulated with CD3+CD28+CD40 overnight then stained for CD4 (bar graph and right histogram; grey tinted – stain-isotype; black line – isotype treated; dotted line – CD3+CD28+CD40 stimulated). (D) NOD CD4loCD40+ T cells were CFSE labeled then isotype treated (Isotype) or CD3-stimulated (CD3XL) in the absence/presence of indicated concentrations of galectin-9 (gal-9) for 8 days. Proliferation was measured by CFSE dilution. All bar graphs in this figure depict means with SEM. Asterisks denote significant differences determined by one- or two-way Anova as appropriate; ns – not significant; * – P between 0.01 and 0.05; ** – P <0.01; *** – P <0.001; **** – P<0.0001. Experiments were done at least three separate times.

Mentions: When culturing CD4loCD40+ T cells in the presence of galectin-9, a change in the morphology was noted. Normally, when those cells are CD40 engaged distinct ball-shaped clusters are formed [17] while CD4hi T cells form more elongated clusters (Fig. 5A). In the presence of galectin-9 the CD4loCD40+ T cells took on a morphology that closely resembled that of CD4hi T cells in culture (Fig. 5A). This led us to determine whether the galectin-9 treated CD4loCD40+ T cells also had a protein surface expression resembling that of CD4hi T cells. CD4loCD40+ T cells have commonly been discarded or gated out as non-T cells due to their low surface expression of CD4, CD3 and TCR but we demonstrated that those cells have high levels of those proteins intracellularly [13]. NOD CD4loCD40+ T cells were treated with sub-lethal levels, 2 5 µg/ml, of galectin-9 then stained for CD4, CD3, TCR and CD5. Those proteins were up-regulated on the treated cells as early as two hours after the treatment (Fig. 5B). A stain for CD8 revealed extremely few, 1–2%, CD8+ cells confirming that the CD4loCD40+ T cells conform to the CD4 lineage.


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 causes autoimmune CD4loCD40+ T cells to appear more like CD4hi T cells and increases CD3 induced proliferation.CD4loCD40+ and CD4hi T cells were sorted from 7–13 weeks old female NOD spleens. (A) CD4loCD40+ T cells were isotype treated (Isotype) or CD40-stimulated for 2 days in the absence/presence of indicated concentrations of galectin-9 then photographed under a microscope. (B) NOD CD4loCD40+ T cells were treated for 2 hours with 2.5 µg/ml galectin-9 then stained for CD3, CD4, TCR, CD5 and CD8. Tinted – untreated; black line – 2.5 µg/ml galectin-9. Gates were set based on appropriate isotype controls. Below each histogram is a corresponding bar graph depicting the cumulative data. (C) NOD CD4hi T cells were either stained immediately for CD28 or CD40 (left two histograms; grey tinted – stain-isotype; black line – CD28 or CD40 as indicated in figure; percentages are means with SEM) or isotype treated or stimulated with CD3+CD28+CD40 overnight then stained for CD4 (bar graph and right histogram; grey tinted – stain-isotype; black line – isotype treated; dotted line – CD3+CD28+CD40 stimulated). (D) NOD CD4loCD40+ T cells were CFSE labeled then isotype treated (Isotype) or CD3-stimulated (CD3XL) in the absence/presence of indicated concentrations of galectin-9 (gal-9) for 8 days. Proliferation was measured by CFSE dilution. All bar graphs in this figure depict means with SEM. Asterisks denote significant differences determined by one- or two-way Anova as appropriate; ns – not significant; * – P between 0.01 and 0.05; ** – P <0.01; *** – P <0.001; **** – P<0.0001. Experiments were done at least three separate times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369903&req=5

pone-0038708-g005: Galectin-9 causes autoimmune CD4loCD40+ T cells to appear more like CD4hi T cells and increases CD3 induced proliferation.CD4loCD40+ and CD4hi T cells were sorted from 7–13 weeks old female NOD spleens. (A) CD4loCD40+ T cells were isotype treated (Isotype) or CD40-stimulated for 2 days in the absence/presence of indicated concentrations of galectin-9 then photographed under a microscope. (B) NOD CD4loCD40+ T cells were treated for 2 hours with 2.5 µg/ml galectin-9 then stained for CD3, CD4, TCR, CD5 and CD8. Tinted – untreated; black line – 2.5 µg/ml galectin-9. Gates were set based on appropriate isotype controls. Below each histogram is a corresponding bar graph depicting the cumulative data. (C) NOD CD4hi T cells were either stained immediately for CD28 or CD40 (left two histograms; grey tinted – stain-isotype; black line – CD28 or CD40 as indicated in figure; percentages are means with SEM) or isotype treated or stimulated with CD3+CD28+CD40 overnight then stained for CD4 (bar graph and right histogram; grey tinted – stain-isotype; black line – isotype treated; dotted line – CD3+CD28+CD40 stimulated). (D) NOD CD4loCD40+ T cells were CFSE labeled then isotype treated (Isotype) or CD3-stimulated (CD3XL) in the absence/presence of indicated concentrations of galectin-9 (gal-9) for 8 days. Proliferation was measured by CFSE dilution. All bar graphs in this figure depict means with SEM. Asterisks denote significant differences determined by one- or two-way Anova as appropriate; ns – not significant; * – P between 0.01 and 0.05; ** – P <0.01; *** – P <0.001; **** – P<0.0001. Experiments were done at least three separate times.
Mentions: When culturing CD4loCD40+ T cells in the presence of galectin-9, a change in the morphology was noted. Normally, when those cells are CD40 engaged distinct ball-shaped clusters are formed [17] while CD4hi T cells form more elongated clusters (Fig. 5A). In the presence of galectin-9 the CD4loCD40+ T cells took on a morphology that closely resembled that of CD4hi T cells in culture (Fig. 5A). This led us to determine whether the galectin-9 treated CD4loCD40+ T cells also had a protein surface expression resembling that of CD4hi T cells. CD4loCD40+ T cells have commonly been discarded or gated out as non-T cells due to their low surface expression of CD4, CD3 and TCR but we demonstrated that those cells have high levels of those proteins intracellularly [13]. NOD CD4loCD40+ T cells were treated with sub-lethal levels, 2 5 µg/ml, of galectin-9 then stained for CD4, CD3, TCR and CD5. Those proteins were up-regulated on the treated cells as early as two hours after the treatment (Fig. 5B). A stain for CD8 revealed extremely few, 1–2%, CD8+ cells confirming that the CD4loCD40+ T cells conform to the CD4 lineage.

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