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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 interacts with CD40.CD4loCD40+ T cells were sorted from female NOD, control BALB/c (BALB con.), or agonistic CD40 antibody injected BALB/c (BALB exp.) spleens. Whole cell lysates were prepared. (A) CD40 was immunoprecipitated and resulting proteins were separated by SDS-PAGE. Protein bands were sequenced by LC-LC-MS. (B) Cells were CD40 stimulated (CD40XL) for 0, 30 minutes, 3 hours or overnight (0′, 30′, 3h, o.n.) then lysates were prepared and CD40 immunoprecipitated. Co-imunoprecipitation of galectin-9 was measured in western blots. Data represents three separate experiments on mice ranging from 8 – 10 weeks old. (C) Cells were isotype or CD40 stimulated (CD40XL) overnight then lysates were prepared. Galectin-9 protein conjugated directly to magnetic beads was used in immunoprecipitations. Mock treated beads were used as a control. CD40 was measured in the immunoprecipitated material by western blot. Data represents three separate mice of two different ages (8 and 10 weeks old).
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pone-0038708-g004: Galectin-9 interacts with CD40.CD4loCD40+ T cells were sorted from female NOD, control BALB/c (BALB con.), or agonistic CD40 antibody injected BALB/c (BALB exp.) spleens. Whole cell lysates were prepared. (A) CD40 was immunoprecipitated and resulting proteins were separated by SDS-PAGE. Protein bands were sequenced by LC-LC-MS. (B) Cells were CD40 stimulated (CD40XL) for 0, 30 minutes, 3 hours or overnight (0′, 30′, 3h, o.n.) then lysates were prepared and CD40 immunoprecipitated. Co-imunoprecipitation of galectin-9 was measured in western blots. Data represents three separate experiments on mice ranging from 8 – 10 weeks old. (C) Cells were isotype or CD40 stimulated (CD40XL) overnight then lysates were prepared. Galectin-9 protein conjugated directly to magnetic beads was used in immunoprecipitations. Mock treated beads were used as a control. CD40 was measured in the immunoprecipitated material by western blot. Data represents three separate mice of two different ages (8 and 10 weeks old).

Mentions: Because galectin-9 had a profound impact on CD40 induced survival and proliferation of CD4loCD40+ T cells we determined whether galectin-9 could directly interact with CD40. We performed CD40 co-immunoprecipitation on NOD CD4loCD40+ T cell whole cell extracts and separated the resulting proteins on polyacrylamide gels followed by LC-LC-MS identification. Several proteins were identified with galectin-9 being one of them (Fig. 4A). These cells had no exogenously added galectin-9 and therefore the co-immunoprecipitated galectin-9 represents endogenous galectin-9. Other proteins of specific interest are VDAC1 (Voltage-Dependent Anion-selective Channel protein) and VDAC3. CD40 induces high levels of Bcl-XL in NOD CD4loCD40+ T cells [13] and VDAC1 and VDAC3 are proteins found in the mitochondrial outer membrane where they interact with proteins such as Bcl-XL to prevent the release of apoptogenic proteins [30]. To corroborate the galectin-9 protein sequencing data we performed CD40 co-immunoprecipitation followed by western blot for galectin-9 on both NOD and BALB/c CD4loCD40+ T cells. While there was a weak interaction between CD40 and galectin-9 in NOD CD4loCD40+ T cells, that interaction was much stronger in control BALB/c CD4loCD40+ T cells (Fig. 4 B). When in-vivo expanded BALB/c CD4loCD40+ T cells were examined the interaction was less strong at earlier time points of CD40 engagement but became stronger with longer time (Fig. 4B). Although the isotype immunoprecipitation did not yield any galectin-9, since galectins technically could interact with carbohydrates on an antibody we performed co-immunoprecipitations utilizing galectin-9 protein immobilized onto magnetic microbeads. This co-immunoprecipitation strategy revealed CD40 protein in a western blot (Fig. 4C) demonstrating again that the two proteins interact directly.


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 interacts with CD40.CD4loCD40+ T cells were sorted from female NOD, control BALB/c (BALB con.), or agonistic CD40 antibody injected BALB/c (BALB exp.) spleens. Whole cell lysates were prepared. (A) CD40 was immunoprecipitated and resulting proteins were separated by SDS-PAGE. Protein bands were sequenced by LC-LC-MS. (B) Cells were CD40 stimulated (CD40XL) for 0, 30 minutes, 3 hours or overnight (0′, 30′, 3h, o.n.) then lysates were prepared and CD40 immunoprecipitated. Co-imunoprecipitation of galectin-9 was measured in western blots. Data represents three separate experiments on mice ranging from 8 – 10 weeks old. (C) Cells were isotype or CD40 stimulated (CD40XL) overnight then lysates were prepared. Galectin-9 protein conjugated directly to magnetic beads was used in immunoprecipitations. Mock treated beads were used as a control. CD40 was measured in the immunoprecipitated material by western blot. Data represents three separate mice of two different ages (8 and 10 weeks old).
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Related In: Results  -  Collection

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pone-0038708-g004: Galectin-9 interacts with CD40.CD4loCD40+ T cells were sorted from female NOD, control BALB/c (BALB con.), or agonistic CD40 antibody injected BALB/c (BALB exp.) spleens. Whole cell lysates were prepared. (A) CD40 was immunoprecipitated and resulting proteins were separated by SDS-PAGE. Protein bands were sequenced by LC-LC-MS. (B) Cells were CD40 stimulated (CD40XL) for 0, 30 minutes, 3 hours or overnight (0′, 30′, 3h, o.n.) then lysates were prepared and CD40 immunoprecipitated. Co-imunoprecipitation of galectin-9 was measured in western blots. Data represents three separate experiments on mice ranging from 8 – 10 weeks old. (C) Cells were isotype or CD40 stimulated (CD40XL) overnight then lysates were prepared. Galectin-9 protein conjugated directly to magnetic beads was used in immunoprecipitations. Mock treated beads were used as a control. CD40 was measured in the immunoprecipitated material by western blot. Data represents three separate mice of two different ages (8 and 10 weeks old).
Mentions: Because galectin-9 had a profound impact on CD40 induced survival and proliferation of CD4loCD40+ T cells we determined whether galectin-9 could directly interact with CD40. We performed CD40 co-immunoprecipitation on NOD CD4loCD40+ T cell whole cell extracts and separated the resulting proteins on polyacrylamide gels followed by LC-LC-MS identification. Several proteins were identified with galectin-9 being one of them (Fig. 4A). These cells had no exogenously added galectin-9 and therefore the co-immunoprecipitated galectin-9 represents endogenous galectin-9. Other proteins of specific interest are VDAC1 (Voltage-Dependent Anion-selective Channel protein) and VDAC3. CD40 induces high levels of Bcl-XL in NOD CD4loCD40+ T cells [13] and VDAC1 and VDAC3 are proteins found in the mitochondrial outer membrane where they interact with proteins such as Bcl-XL to prevent the release of apoptogenic proteins [30]. To corroborate the galectin-9 protein sequencing data we performed CD40 co-immunoprecipitation followed by western blot for galectin-9 on both NOD and BALB/c CD4loCD40+ T cells. While there was a weak interaction between CD40 and galectin-9 in NOD CD4loCD40+ T cells, that interaction was much stronger in control BALB/c CD4loCD40+ T cells (Fig. 4 B). When in-vivo expanded BALB/c CD4loCD40+ T cells were examined the interaction was less strong at earlier time points of CD40 engagement but became stronger with longer time (Fig. 4B). Although the isotype immunoprecipitation did not yield any galectin-9, since galectins technically could interact with carbohydrates on an antibody we performed co-immunoprecipitations utilizing galectin-9 protein immobilized onto magnetic microbeads. This co-immunoprecipitation strategy revealed CD40 protein in a western blot (Fig. 4C) demonstrating again that the two proteins interact directly.

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