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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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Related in: MedlinePlus

Galectin-9 prevents CD40 induced proliferation.(A) CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were then labeled with CFSE. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of increasing concentrations of galectin-9 (gal-9) for 4 days then CFSE dilution was measured. (B) Cells were sorted and treated as in A and were then stained for necrotic and apoptotic cell death. (C) Cells were sorted and CD40 stimulated as in A for 24 hours then galectin-9 was added at 7.5 µg/ml. Cells were analyzed for proliferation and cell death after a total of 4 days of stimulation. (D and E) Cells were sorted as in A and were pretreated, or not, with a Tim-3 blocking antibody (αTim-3) for 30 miutes then CD40 was stimulated in the absence/presence of galectin-9 for 4 days. Proliferation (D) and cell death (E) was measured, respectively. Percentages in A are means with SEM. Asterisks in B, C, and D denote significant differences determined by two-way Anova; 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.
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pone-0038708-g001: Galectin-9 prevents CD40 induced proliferation.(A) CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were then labeled with CFSE. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of increasing concentrations of galectin-9 (gal-9) for 4 days then CFSE dilution was measured. (B) Cells were sorted and treated as in A and were then stained for necrotic and apoptotic cell death. (C) Cells were sorted and CD40 stimulated as in A for 24 hours then galectin-9 was added at 7.5 µg/ml. Cells were analyzed for proliferation and cell death after a total of 4 days of stimulation. (D and E) Cells were sorted as in A and were pretreated, or not, with a Tim-3 blocking antibody (αTim-3) for 30 miutes then CD40 was stimulated in the absence/presence of galectin-9 for 4 days. Proliferation (D) and cell death (E) was measured, respectively. Percentages in A are means with SEM. Asterisks in B, C, and D denote significant differences determined by two-way Anova; 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: Expansion of CD4loCD40+ T cells can be prevented by blocking CD40 – CD154 interactions [14], [17]. To date little is known about how to control CD4loCD40+ T cells after they are expanded and activated. We demonstrated that engagement of TNFR1 and/or TNFR2 in addition to CD40 prevented CD40 induced proliferation of autoimmune NOD CD4loCD40+ T cells [17] as did Fas engagement [13]. However, those treatments did not kill the CD4loCD40+ T cells. Because galectin-9 has been shown to induce cell death in Th1 cells [27] we determined whether it could also affect NOD CD4loCD40+ T cells. Galectin-9 prevented CD40 induced proliferation in a dose dependent manner (Fig. 1A) and induced necrotic cell death in the CD40 stimulated cells (Fig. 1B; left bar graph). Cells that were isotype treated for four days underwent necrotic cell death and a large portion of that was prevented by CD40-stimulation (Fig. 1B; 49.5% and 26.3% cell death respectively). Addition of a lower concentration, 2.5 µg/ml, of galectin-9 did not prevent the CD40 induced survival (Fig. 1B; 29% cell death), however, when higher concentrations were added much of the CD40 induced survival was prevented. Cell death rates were confirmed with absolute cell counts in trypan blue (data not shown). Apoptotic cell death was not apparent in the CD4loCD40+ T cells (Fig. 1B; right bar graph).


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 prevents CD40 induced proliferation.(A) CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were then labeled with CFSE. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of increasing concentrations of galectin-9 (gal-9) for 4 days then CFSE dilution was measured. (B) Cells were sorted and treated as in A and were then stained for necrotic and apoptotic cell death. (C) Cells were sorted and CD40 stimulated as in A for 24 hours then galectin-9 was added at 7.5 µg/ml. Cells were analyzed for proliferation and cell death after a total of 4 days of stimulation. (D and E) Cells were sorted as in A and were pretreated, or not, with a Tim-3 blocking antibody (αTim-3) for 30 miutes then CD40 was stimulated in the absence/presence of galectin-9 for 4 days. Proliferation (D) and cell death (E) was measured, respectively. Percentages in A are means with SEM. Asterisks in B, C, and D denote significant differences determined by two-way Anova; 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

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getmorefigures.php?uid=PMC3369903&req=5

pone-0038708-g001: Galectin-9 prevents CD40 induced proliferation.(A) CD4loCD40+ T cells were sorted from 7–13 week old female NOD spleens and were then labeled with CFSE. Cells were either isotype treated (Isotype) or CD40 was stimulated (CD40XL) in the absence/presence of increasing concentrations of galectin-9 (gal-9) for 4 days then CFSE dilution was measured. (B) Cells were sorted and treated as in A and were then stained for necrotic and apoptotic cell death. (C) Cells were sorted and CD40 stimulated as in A for 24 hours then galectin-9 was added at 7.5 µg/ml. Cells were analyzed for proliferation and cell death after a total of 4 days of stimulation. (D and E) Cells were sorted as in A and were pretreated, or not, with a Tim-3 blocking antibody (αTim-3) for 30 miutes then CD40 was stimulated in the absence/presence of galectin-9 for 4 days. Proliferation (D) and cell death (E) was measured, respectively. Percentages in A are means with SEM. Asterisks in B, C, and D denote significant differences determined by two-way Anova; 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: Expansion of CD4loCD40+ T cells can be prevented by blocking CD40 – CD154 interactions [14], [17]. To date little is known about how to control CD4loCD40+ T cells after they are expanded and activated. We demonstrated that engagement of TNFR1 and/or TNFR2 in addition to CD40 prevented CD40 induced proliferation of autoimmune NOD CD4loCD40+ T cells [17] as did Fas engagement [13]. However, those treatments did not kill the CD4loCD40+ T cells. Because galectin-9 has been shown to induce cell death in Th1 cells [27] we determined whether it could also affect NOD CD4loCD40+ T cells. Galectin-9 prevented CD40 induced proliferation in a dose dependent manner (Fig. 1A) and induced necrotic cell death in the CD40 stimulated cells (Fig. 1B; left bar graph). Cells that were isotype treated for four days underwent necrotic cell death and a large portion of that was prevented by CD40-stimulation (Fig. 1B; 49.5% and 26.3% cell death respectively). Addition of a lower concentration, 2.5 µg/ml, of galectin-9 did not prevent the CD40 induced survival (Fig. 1B; 29% cell death), however, when higher concentrations were added much of the CD40 induced survival was prevented. Cell death rates were confirmed with absolute cell counts in trypan blue (data not shown). Apoptotic cell death was not apparent in the CD4loCD40+ T cells (Fig. 1B; right bar graph).

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