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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

In-vivo CD40 signals cause non-autoimmune CD4loCD40+ T cells to appear more like autoimmune CD4loCD40+ T cells.(A) 10–12 week old, female BALB/c mice were injected i.p. with 1C10+FGK45 agonistic CD40 antibodies (CD40-inj.) or with isotype control (control) antibody. Three days post-injection, splenic CD4loCD40+ and CD4hi T cells were purified and stained with L-PHA. Graph depicts the difference in L-PHA stain compared to control. Data are represented as means with SEM. Images are H&E stained sections of pancreata. (B) BALB/c mice were treated and CD4loCD40+ T cells purified as in A. CD4loCD40+ T cells from 10–12 weeks old female NOD spleens were purified for comparison. Cells were CFSE labeled then CD40-stimulated for 4 days. CFSE dilution was measured. Dotted line – isotype treated; solid line – CD40-stimunlated. (C) 10 week old female BALB/c mice were injected and CD4loCD40+ T cells purified as in A, then cells were labeled with CFSE and isotype treated (Isotype) or CD40 stimulated (CD40XL) in the absence/presence of galectin-9. CFSE dilution was measured. Percentages in B and C are means +/− SEM. Experiments were performed at least three separate times.
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pone-0038708-g003: In-vivo CD40 signals cause non-autoimmune CD4loCD40+ T cells to appear more like autoimmune CD4loCD40+ T cells.(A) 10–12 week old, female BALB/c mice were injected i.p. with 1C10+FGK45 agonistic CD40 antibodies (CD40-inj.) or with isotype control (control) antibody. Three days post-injection, splenic CD4loCD40+ and CD4hi T cells were purified and stained with L-PHA. Graph depicts the difference in L-PHA stain compared to control. Data are represented as means with SEM. Images are H&E stained sections of pancreata. (B) BALB/c mice were treated and CD4loCD40+ T cells purified as in A. CD4loCD40+ T cells from 10–12 weeks old female NOD spleens were purified for comparison. Cells were CFSE labeled then CD40-stimulated for 4 days. CFSE dilution was measured. Dotted line – isotype treated; solid line – CD40-stimunlated. (C) 10 week old female BALB/c mice were injected and CD4loCD40+ T cells purified as in A, then cells were labeled with CFSE and isotype treated (Isotype) or CD40 stimulated (CD40XL) in the absence/presence of galectin-9. CFSE dilution was measured. Percentages in B and C are means +/− SEM. Experiments were performed at least three separate times.

Mentions: We previously demonstrated that injection of non-autoimmune BALB/c mice with agonistic CD40 antibodies expanded the CD4loCD40+ T cell population after three to six days to numbers seen in autoimmune NOD mice [17]. Since that treatment also altered the CD40 glycoform profile in those cells we determined whether CD40 signals had an impact on glycosylation status beyond just the CD40 protein itself in CD4 T cells. The extent of β1,6GlcNAc-branched N-glycans was assayed using L-PHA, a plant lectin that binds to carbohydrate structures on glycoproteins that are also recognized by galectins [23]. Injection of agonistic CD40 antibodies into non-autoimmune BALB/c mice caused a reduction in β1,6GlcNAc-branched N-glycans on CD4loCD40+ and CD4hi T cells to a level similar to that of autoimmune NOD mice [23] (Fig. 3A; bar graph). This then could have a direct impact on the activation status of those cells as has been shown [23]. After fourteen days, the splenic expansion of CD4loCD40+ T cells in the BALB/c mice receded to normal numbers (unpublished data). Interestingly, in response to the in-vivo CD40 stimulation, the pancreatic lymph nodes became populated while in control mice no populated nodes were found (Fig. 3A; H&E stained images). CD4loCD40+ T cells from control BALB/c mice do not proliferate in response to CD40 stimulation in-vitro[13]. We determined whether the expanded CD4loCD40+ T cells had acquired the ability to proliferate in response to CD40 in-vitro in the same manner as NOD CD4loCD40+ T cells do [13] (Fig. 1A). CD4loCD40+ T cells purified three days after a single injection with agonistic CD40 antibodies readily proliferated in response to CD40 engagement in-vitro (Fig. 3B). However, the expanded BALB/c CD4loCD40+ T cells did not proliferate to the same extent as NOD CD4loCD40+ T cells; 45% of BALB/c CD4loCD40+ T cells proliferated compared to 67.8% of NOD CD4loCD40+ T cells (Fig. 3B). As CD4loCD40+ T cells from untreated BALB/c mice do not proliferate in-vitro in response to CD40 engagement [13] and since expanded BALB/c CD4loCD40+ T cells display a CD40 glycoform profile similar to NOD CD4loCD40+ T cells [17], the new data indicates that the acquisition of the ‘correct’ CD40 glycoform profile is required before induction of proliferation through CD40 in-vitro is possible.


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)

In-vivo CD40 signals cause non-autoimmune CD4loCD40+ T cells to appear more like autoimmune CD4loCD40+ T cells.(A) 10–12 week old, female BALB/c mice were injected i.p. with 1C10+FGK45 agonistic CD40 antibodies (CD40-inj.) or with isotype control (control) antibody. Three days post-injection, splenic CD4loCD40+ and CD4hi T cells were purified and stained with L-PHA. Graph depicts the difference in L-PHA stain compared to control. Data are represented as means with SEM. Images are H&E stained sections of pancreata. (B) BALB/c mice were treated and CD4loCD40+ T cells purified as in A. CD4loCD40+ T cells from 10–12 weeks old female NOD spleens were purified for comparison. Cells were CFSE labeled then CD40-stimulated for 4 days. CFSE dilution was measured. Dotted line – isotype treated; solid line – CD40-stimunlated. (C) 10 week old female BALB/c mice were injected and CD4loCD40+ T cells purified as in A, then cells were labeled with CFSE and isotype treated (Isotype) or CD40 stimulated (CD40XL) in the absence/presence of galectin-9. CFSE dilution was measured. Percentages in B and C are means +/− SEM. Experiments were performed at least three separate times.
© Copyright Policy
Related In: Results  -  Collection

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pone-0038708-g003: In-vivo CD40 signals cause non-autoimmune CD4loCD40+ T cells to appear more like autoimmune CD4loCD40+ T cells.(A) 10–12 week old, female BALB/c mice were injected i.p. with 1C10+FGK45 agonistic CD40 antibodies (CD40-inj.) or with isotype control (control) antibody. Three days post-injection, splenic CD4loCD40+ and CD4hi T cells were purified and stained with L-PHA. Graph depicts the difference in L-PHA stain compared to control. Data are represented as means with SEM. Images are H&E stained sections of pancreata. (B) BALB/c mice were treated and CD4loCD40+ T cells purified as in A. CD4loCD40+ T cells from 10–12 weeks old female NOD spleens were purified for comparison. Cells were CFSE labeled then CD40-stimulated for 4 days. CFSE dilution was measured. Dotted line – isotype treated; solid line – CD40-stimunlated. (C) 10 week old female BALB/c mice were injected and CD4loCD40+ T cells purified as in A, then cells were labeled with CFSE and isotype treated (Isotype) or CD40 stimulated (CD40XL) in the absence/presence of galectin-9. CFSE dilution was measured. Percentages in B and C are means +/− SEM. Experiments were performed at least three separate times.
Mentions: We previously demonstrated that injection of non-autoimmune BALB/c mice with agonistic CD40 antibodies expanded the CD4loCD40+ T cell population after three to six days to numbers seen in autoimmune NOD mice [17]. Since that treatment also altered the CD40 glycoform profile in those cells we determined whether CD40 signals had an impact on glycosylation status beyond just the CD40 protein itself in CD4 T cells. The extent of β1,6GlcNAc-branched N-glycans was assayed using L-PHA, a plant lectin that binds to carbohydrate structures on glycoproteins that are also recognized by galectins [23]. Injection of agonistic CD40 antibodies into non-autoimmune BALB/c mice caused a reduction in β1,6GlcNAc-branched N-glycans on CD4loCD40+ and CD4hi T cells to a level similar to that of autoimmune NOD mice [23] (Fig. 3A; bar graph). This then could have a direct impact on the activation status of those cells as has been shown [23]. After fourteen days, the splenic expansion of CD4loCD40+ T cells in the BALB/c mice receded to normal numbers (unpublished data). Interestingly, in response to the in-vivo CD40 stimulation, the pancreatic lymph nodes became populated while in control mice no populated nodes were found (Fig. 3A; H&E stained images). CD4loCD40+ T cells from control BALB/c mice do not proliferate in response to CD40 stimulation in-vitro[13]. We determined whether the expanded CD4loCD40+ T cells had acquired the ability to proliferate in response to CD40 in-vitro in the same manner as NOD CD4loCD40+ T cells do [13] (Fig. 1A). CD4loCD40+ T cells purified three days after a single injection with agonistic CD40 antibodies readily proliferated in response to CD40 engagement in-vitro (Fig. 3B). However, the expanded BALB/c CD4loCD40+ T cells did not proliferate to the same extent as NOD CD4loCD40+ T cells; 45% of BALB/c CD4loCD40+ T cells proliferated compared to 67.8% of NOD CD4loCD40+ T cells (Fig. 3B). As CD4loCD40+ T cells from untreated BALB/c mice do not proliferate in-vitro in response to CD40 engagement [13] and since expanded BALB/c CD4loCD40+ T cells display a CD40 glycoform profile similar to NOD CD4loCD40+ T cells [17], the new data indicates that the acquisition of the ‘correct’ CD40 glycoform profile is required before induction of proliferation through CD40 in-vitro is possible.

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