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Galectin-9-mediated protection from allo-specific T cells as a mechanism of immune privilege of corneal allografts.

Shimmura-Tomita M, Wang M, Taniguchi H, Akiba H, Yagita H, Hori J - PLoS ONE (2013)

Bottom Line: The eye is an immune-privileged organ, and corneal transplantation is therefore one of the most successful organ transplantation.T-cell immunoglobulin and mucin domain (Tim)-3 is a regulatory molecule for T-cell function, and galectin (Gal)-9 is a Tim-3 ligand.Blockade of Tim-3 or Gal-9 did not abolish anterior chamber-associated immune deviation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan. machiko@jichi.ac.jp

ABSTRACT
The eye is an immune-privileged organ, and corneal transplantation is therefore one of the most successful organ transplantation. The immunosuppressive intraocular microenvironment is known as one of the mechanisms underlying immune privilege in the eye. T-cell immunoglobulin and mucin domain (Tim)-3 is a regulatory molecule for T-cell function, and galectin (Gal)-9 is a Tim-3 ligand. We investigated the role of this pathway in establishing the immune-privileged status of corneal allografts in mice. Gal-9 is constitutively expressed on the corneal epithelium, endothelium and iris-ciliary body in normal mouse eyes and eyes bearing surviving allografts, and Tim-3 was expressed on CD8 T cells infiltrating the allografts. Allograft survival in recipients treated with anti-Tim-3 monoclonal antibody (mAb) or anti-Gal-9 mAb was significantly shorter than that in control recipients. In vitro, destruction of corneal endothelial cells by allo-reactive T cells was enhanced when the cornea was pretreated with anti-Gal-9 mAb. Blockade of Tim-3 or Gal-9 did not abolish anterior chamber-associated immune deviation. We propose that constitutive expression of Gal-9 plays an immunosuppressive role in corneal allografts. Gal-9 expressed on corneal endothelial cells protects them from destruction by allo-reactive T cells within the cornea.

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Gal-9 protects corneal endothelial cells from killing by alloreactive T cells.(A) C57BL/6 (B6) corneas pretreated with anti-Gal-9 mAb (RG9-35) or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against B6 alloantigens (α-B6/BALB-T cells) or third-party C3H/He alloantigens (α-C3H/BALB-T cells), or from the spleens of naïve BALB/c (naïve BALB-T cells), B6 (naïve B6-T cells), or C3H/He mice (naïve C3H-T cells). After a 6-h incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without Ab treatment or incubation with T cells. As negative controls, corneas with Ab treatment and incubation without T cells (antibody only), and corneas without Ab treatment or incubation with T cells (no antibody no T cells) were used. Data are presented as the mean ± standard error of five corneas in each group. (B, C) Apoptosis of allo-reactive T cells following co-incubation with B6 corneas pre-treated with anti-Gal-9 mAb or control IgG was examined by flow cytometry with FITC-conjugated Annexin V/PI. Apoptosis of CD4+ T cells was significantly suppressed with anti-Gal-9 mAb treatment compared to control, while there was no difference in apoptosis of CD8+ T cells. B shows the representative flow cytometry data. C shows the data presented as the mean ± standard deviation of six experiments in each group.
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pone-0063620-g005: Gal-9 protects corneal endothelial cells from killing by alloreactive T cells.(A) C57BL/6 (B6) corneas pretreated with anti-Gal-9 mAb (RG9-35) or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against B6 alloantigens (α-B6/BALB-T cells) or third-party C3H/He alloantigens (α-C3H/BALB-T cells), or from the spleens of naïve BALB/c (naïve BALB-T cells), B6 (naïve B6-T cells), or C3H/He mice (naïve C3H-T cells). After a 6-h incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without Ab treatment or incubation with T cells. As negative controls, corneas with Ab treatment and incubation without T cells (antibody only), and corneas without Ab treatment or incubation with T cells (no antibody no T cells) were used. Data are presented as the mean ± standard error of five corneas in each group. (B, C) Apoptosis of allo-reactive T cells following co-incubation with B6 corneas pre-treated with anti-Gal-9 mAb or control IgG was examined by flow cytometry with FITC-conjugated Annexin V/PI. Apoptosis of CD4+ T cells was significantly suppressed with anti-Gal-9 mAb treatment compared to control, while there was no difference in apoptosis of CD8+ T cells. B shows the representative flow cytometry data. C shows the data presented as the mean ± standard deviation of six experiments in each group.

Mentions: The constitutive Gal-9 expression in surviving corneal allografts led us to postulate a hypothesis, in which the Gal-9 molecules have the capacity to protect corneal allografts from alloreactive infiltrating T cells by inducing apoptosis of these effector cells. To address this possibility, we used a model of corneal endothelial cell destruction by alloreactive T cells in vitro that we had established previously [29]. In previous studies by us and others, corneal endothelial cells have been documented to represent a target of alloreactive T cells in both human and rodent corneal transplantations [51]. As a model of the effector phase of corneal rejection, normal C57BL/6 corneas were incubated with purified T cells from the spleens of BALB/c mice presensitized against C57BL/6 alloantigens. Purified T cells from the spleens of BALB/c mice presensitized against third-party C3H/He alloantigens were used as non specifically activated T cells. Splenic T cells from naive BALB/c, C57BL/6, and C3H/He mice were used as allogeneic, syngeneic, and third-party naive T cells, respectively. The number of dead CECs was significantly increased in anti-Gal-9 mAb-treated corneas than in control IgG-treated corneas after incubation with allo-reactive T cells (p = 0.0006) (Fig. 5A). In contrast, no significant difference was observed between anti-Gal-9 mAb- and control IgG-treated corneas when activated T cells against third-party allo-antigens, naïve BALB/c or C3H/He T cells, and syngeneic C57BL/6 T cells were used as the effector cells. These results suggest that Gal-9 protects corneal endothelial cells from killing by allo-reactive T cells. When the co-culture of allo-reactive T cells and corneal endothelial cells were treated with anti-Gal-9 mAb, apoptosis of CD4+ T cells was significantly suppressed compared to control, while there was no difference in apoptosis of CD8+ T cells (Fig. 5B, C, D).


Galectin-9-mediated protection from allo-specific T cells as a mechanism of immune privilege of corneal allografts.

Shimmura-Tomita M, Wang M, Taniguchi H, Akiba H, Yagita H, Hori J - PLoS ONE (2013)

Gal-9 protects corneal endothelial cells from killing by alloreactive T cells.(A) C57BL/6 (B6) corneas pretreated with anti-Gal-9 mAb (RG9-35) or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against B6 alloantigens (α-B6/BALB-T cells) or third-party C3H/He alloantigens (α-C3H/BALB-T cells), or from the spleens of naïve BALB/c (naïve BALB-T cells), B6 (naïve B6-T cells), or C3H/He mice (naïve C3H-T cells). After a 6-h incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without Ab treatment or incubation with T cells. As negative controls, corneas with Ab treatment and incubation without T cells (antibody only), and corneas without Ab treatment or incubation with T cells (no antibody no T cells) were used. Data are presented as the mean ± standard error of five corneas in each group. (B, C) Apoptosis of allo-reactive T cells following co-incubation with B6 corneas pre-treated with anti-Gal-9 mAb or control IgG was examined by flow cytometry with FITC-conjugated Annexin V/PI. Apoptosis of CD4+ T cells was significantly suppressed with anti-Gal-9 mAb treatment compared to control, while there was no difference in apoptosis of CD8+ T cells. B shows the representative flow cytometry data. C shows the data presented as the mean ± standard deviation of six experiments in each group.
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Related In: Results  -  Collection

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pone-0063620-g005: Gal-9 protects corneal endothelial cells from killing by alloreactive T cells.(A) C57BL/6 (B6) corneas pretreated with anti-Gal-9 mAb (RG9-35) or control IgG were incubated with purified T cells from the spleens of BALB/c mice presensitized against B6 alloantigens (α-B6/BALB-T cells) or third-party C3H/He alloantigens (α-C3H/BALB-T cells), or from the spleens of naïve BALB/c (naïve BALB-T cells), B6 (naïve B6-T cells), or C3H/He mice (naïve C3H-T cells). After a 6-h incubation, corneal endothelial cell death was detected by staining unfixed tissue with PI followed by confocal microscopic examination. Positive control corneas were incubated with Triton X-100, without Ab treatment or incubation with T cells. As negative controls, corneas with Ab treatment and incubation without T cells (antibody only), and corneas without Ab treatment or incubation with T cells (no antibody no T cells) were used. Data are presented as the mean ± standard error of five corneas in each group. (B, C) Apoptosis of allo-reactive T cells following co-incubation with B6 corneas pre-treated with anti-Gal-9 mAb or control IgG was examined by flow cytometry with FITC-conjugated Annexin V/PI. Apoptosis of CD4+ T cells was significantly suppressed with anti-Gal-9 mAb treatment compared to control, while there was no difference in apoptosis of CD8+ T cells. B shows the representative flow cytometry data. C shows the data presented as the mean ± standard deviation of six experiments in each group.
Mentions: The constitutive Gal-9 expression in surviving corneal allografts led us to postulate a hypothesis, in which the Gal-9 molecules have the capacity to protect corneal allografts from alloreactive infiltrating T cells by inducing apoptosis of these effector cells. To address this possibility, we used a model of corneal endothelial cell destruction by alloreactive T cells in vitro that we had established previously [29]. In previous studies by us and others, corneal endothelial cells have been documented to represent a target of alloreactive T cells in both human and rodent corneal transplantations [51]. As a model of the effector phase of corneal rejection, normal C57BL/6 corneas were incubated with purified T cells from the spleens of BALB/c mice presensitized against C57BL/6 alloantigens. Purified T cells from the spleens of BALB/c mice presensitized against third-party C3H/He alloantigens were used as non specifically activated T cells. Splenic T cells from naive BALB/c, C57BL/6, and C3H/He mice were used as allogeneic, syngeneic, and third-party naive T cells, respectively. The number of dead CECs was significantly increased in anti-Gal-9 mAb-treated corneas than in control IgG-treated corneas after incubation with allo-reactive T cells (p = 0.0006) (Fig. 5A). In contrast, no significant difference was observed between anti-Gal-9 mAb- and control IgG-treated corneas when activated T cells against third-party allo-antigens, naïve BALB/c or C3H/He T cells, and syngeneic C57BL/6 T cells were used as the effector cells. These results suggest that Gal-9 protects corneal endothelial cells from killing by allo-reactive T cells. When the co-culture of allo-reactive T cells and corneal endothelial cells were treated with anti-Gal-9 mAb, apoptosis of CD4+ T cells was significantly suppressed compared to control, while there was no difference in apoptosis of CD8+ T cells (Fig. 5B, C, D).

Bottom Line: The eye is an immune-privileged organ, and corneal transplantation is therefore one of the most successful organ transplantation.T-cell immunoglobulin and mucin domain (Tim)-3 is a regulatory molecule for T-cell function, and galectin (Gal)-9 is a Tim-3 ligand.Blockade of Tim-3 or Gal-9 did not abolish anterior chamber-associated immune deviation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan. machiko@jichi.ac.jp

ABSTRACT
The eye is an immune-privileged organ, and corneal transplantation is therefore one of the most successful organ transplantation. The immunosuppressive intraocular microenvironment is known as one of the mechanisms underlying immune privilege in the eye. T-cell immunoglobulin and mucin domain (Tim)-3 is a regulatory molecule for T-cell function, and galectin (Gal)-9 is a Tim-3 ligand. We investigated the role of this pathway in establishing the immune-privileged status of corneal allografts in mice. Gal-9 is constitutively expressed on the corneal epithelium, endothelium and iris-ciliary body in normal mouse eyes and eyes bearing surviving allografts, and Tim-3 was expressed on CD8 T cells infiltrating the allografts. Allograft survival in recipients treated with anti-Tim-3 monoclonal antibody (mAb) or anti-Gal-9 mAb was significantly shorter than that in control recipients. In vitro, destruction of corneal endothelial cells by allo-reactive T cells was enhanced when the cornea was pretreated with anti-Gal-9 mAb. Blockade of Tim-3 or Gal-9 did not abolish anterior chamber-associated immune deviation. We propose that constitutive expression of Gal-9 plays an immunosuppressive role in corneal allografts. Gal-9 expressed on corneal endothelial cells protects them from destruction by allo-reactive T cells within the cornea.

Show MeSH
Related in: MedlinePlus