Gal-9 protects corneal endothelial cells from killing by alloreactive T cells.

<p>(<b>A</b>) 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>B, C</b>) 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.</p

Blockade of Tim-3 or Gal-9 does not abolish ACAID.

<p>C57BL/6 spleen cells were used as alloantigens and injected into the right AC of BALB/c normal eyes. Two weeks later, C57BL/6 spleen cells were injected subcutaneously to sensitize the mice. After one more week, a challenge was conducted by injecting C57BL/6 spleen cells into the right ear pinnae of each mouse, and specific ear swelling was measured 24 h later as an indication of DH. Treatments with anti-Tim-3 mAb, anti-Gal-9 mAb (RG9-1 or RG9-35), or control rat IgG were performed starting from the day of AC injection and continuing until the day of ear challenge. DH responses were similarly suppressed in anti-Tim-3, anti-Gal-9, and control IgG-treated groups, with no significant differences observed among them. Positive control mice (Posi.C) received subcutaneous immunization and ear challenge without previous AC injection. Negative control mice (Nega.C) received only the ear challenge without AC injection or immunization. Data are presented as the mean ± standard error of five corneas in each group.</p

Expression of Tim-3 and Gal-9 in normal eye tissue.

<p>(<b>A</b>) mRNA was extracted from freshly isolated cornea, iris-ciliary body, and neural retina of normal mouse eyes, and then reverse transcribed and amplified by PCR. PCR products were electrophoresed in 2% agarose gel and visualized by ethidium bromide staining. (<b>B</b>) Section of normal mouse cornea was stained using hematoxylin and eosin (HE). (<b>C</b>) Section of normal mouse iris-ciliary body was stained using HE. (<b>D and F</b>) Cryostat sections were stained with anti-Gal-9 mAb followed by FITC-conjugated anti-rat IgG (green). Nuclei were stained with PI (red). (<b>E and G</b>) Cryostat sections were stained with biotinylated anti-Tim-3 Ab followed by streptavidin-FITC (green). Nuclei were stained with DAPI (blue). Ced, corneal endothelium; Cst, corneal stroma; and Cep, corneal epithelium. Original magnification, ×40.</p

Blockade of Gal-9 or Tim-3 increases CD4 and CD8 T cell infiltration in allografts.

<p>Normal corneas of C57BL/6 mice were transplanted into normal eyes of BALB/c mice. All recipients were treated with anti-Tim-3 mAb, anti-Gal-9 mAb, or control rat IgG. Graft-bearing eyes were isolated at 4 weeks. (<b>A, B, D, E, G, and H</b>) Cryostat sections of graft-bearing eyes were examined by HE staining. (<b>C, F, and I</b>) Cryostat sections of graft-bearing eyes were examined by immunofluorescent staining with PE-conjugated anti-CD4 mAb (red) and FITC-conjugated anti-CD8 mAb (green). Nuclei were stained with DAPI (blue). Arrow heads in F and I show CD4+ cells and arrows in F and I show CD8+ cells. Original magnification, ×40. (<b>J, K, L, and M</b>) The number of CD4+ and CD8+ cells at graft center and host-graft junction in recipients treated with anti-Tim-3 mAb and anti-Gal-9 mAb was greater as compared to control recipients. Data are presented as the mean ± standard deviation of four corneas in each group.</p

Expression of Tim-3 and Gal-9 in corneal allografts and secondary lymphoid organs.

<p>Normal corneas of C57BL/6 mice were transplanted into normal eyes of BALB/c mice. Graft-bearing eyes were isolated at 2, 4, and 8 weeks. (<b>A, C, and E</b>) Cryostat sections of surviving graft centers were examined by immunofluorescent staining with anti-Gal-9 mAb followed by FITC-conjugated anti-rat IgG mAb or streptavidin-FITC (green). Nuclei were stained with PI (red). (<b>B, D, F, and G</b>) Cryostat sections of surviving graft centers were examined by immunofluorescent staining with biotinylated anti-Tim-3 Ab followed by streptavidin-FITC (green), and PE-conjugated anti-CD8 mAb (red). Nuclei were stained with DAPI (blue). Arrows in D show the Tim-3+ CD8+ cells, and G represents the magnification of one of the double positive cells. Cep, corneal epithelium; Cst, corneal stroma; Ced, corneal endothelium. Original magnification, ×40. (<b>H, I, J, and K</b>) Whole spleen (SP) or LN cells from recipients bearing surviving (Allo Sur) or rejected (Allo Rej) allografts and syngeneic (Syn) grafts were analyzed by flow cytometry. Data are displayed as the proportion of Tim-3+ CD4+ cells in the whole SP and LN (H, I), Tim-3+ CD8+ cells in the whole SP and LN (J, K). Data are presented as the mean ± standard deviation of five recipients in each group. N.S., not significant.</p

Blockade of Tim-3 or Gal-9 accelerates corneal allograft rejection.

<p>Normal corneas of C57BL/6 mice were transplanted orthotopically into normal eyes of BALB/c mice. After the grafting procedure, recipients were intraperitoneally injected with 0.2 mg of anti-Tim-3 mAb, anti-Gal-9 mAb (RG9-1 or RG9-35), or control rat IgG three times a week for 8 weeks. Graft survival was clinically assessed and compared. Survival of allografts treated with anti-Tim-3 or anti-Gal-9 mAb was significantly shorter than that in control allografts (<i>n</i> = 9–10 in each group).</p

Effect of GM-CSF on the uNK cells and uMDSC from pregnant mice.

<p>A. CBA/CaJ female mice were mated with C57BL/6 allogeneic male mice. Uterine NK cells were flow sorted to obtain a NK1.1^- DX5^- DBA⁺ cell population that was incubated with GMCSF (20ng/ml) ± IL-2 (20ng/ml) for 5–7 days. Expression of uNK and CD11b⁺Ly6G^hiLy6C^lo and CD11b⁺Ly6G^loLy6C^hi cell populations were analyzed using a flow cytometer. GMCSF treatment resulted in no significant change in the NK1.1^- DX5^- DBA⁺ cell population from the CBA mice (p = 0.1164). However, an increase of the CD11b⁺Ly6G^loLy6C^hi population was observed (p = 0.0592). An inverse correlation was observed between the uNK cells and uMDSC in presence of both GMCSF and IL-2 treatment but was not statistically significant. B. C57BL/6 female mice were mated with allogeneic CBA/CaJ male mice. Uterine NK cells were flow sorted to obtain a NK1.1⁺ DX5^- DBA⁺ cell population that was incubated with GMCSF (20ng/ml) ± IL-2 (20ng/ml) for 5–7 days. Expression of uNK and CD11b⁺Ly6G^hiLy6C^lo and CD11b⁺Ly6G^loLy6C^hi cell populations were analyzed using a flow cytometer. GMCSF treatment resulted in a significant change in the NK1.1⁺ DX5^- DBA⁺ cell population from the C57BL/6 female mice (p = 0.0310). However, the change in the CD11b⁺Ly6G^loLy6C^hi population was not significant (p = 0.2410). The effect of GMCSF was more pronounced in the uNK cells from the C57BL/6 female mice and a significant inverse correlation between uNK and uMDSC was also observed (p = 0.0158). Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done by one-way analysis of variance (ANOVA) using Kruskal-Wallis test and two way ANOVA.</p

Expression of CD11b, Ly6C and Ly6G on uNK cells from CBA mice.

<p>Female CBA mice were allogeneically mated toC57BL/6 males and uterine lymphocytes were isolated between GD 10.5 to 12.5. Expression of CD11b, Ly6C and Ly6G was determined on the NK1.1^- DX5^-DBA⁺ cell population. Data are representative of at least 4 experiments.</p

Effect of RMT3-23 treatment on cytokine production by uNK cells.

<p>Treatment with RMT3-23 significantly down regulated cytokine production by NK1.1^- DX5^- DBA⁺ cell population. A significant decrease in the population of IFN, IL-6, IL-10, VEGF and GMCSF producing NK1.1^- DX5^- DBA⁺ cells was observed after treatment with RMT3-23 in comparison to Control group, whereas IL-4 production was significantly increased. Data are presented as mean± SEM and are representative of at least 3 experiments. Statistical analysis was done using unpaired t test.</p

Effect of GM-CSF and IL-15 on the uNK cells from pregnant CBA mice.

<p>CBA/CaJ female mice were mated with C57BL/6 allogeneic male mice. Uterine lymphocytes were isolated from Control and RMT3-23 treated mice and cultured with IL-15, IL-15+IL-12, IL-12, IL-15+GMCSF and GMCSF only for 7days in vitro in a 37°C humidified CO2 incubator. At the end of the incubation period the percentage of 3 different cell populations were determined by flow cytometry. A. RMT3-23 treated uNK cells proliferated more in response to IL-12 (p = 0.0284). B. This effect was more pronounced in the uNK cells expressing the MDSC markers (p<0.0001).</p