Local and Systemic Injections of Human Cord Blood Myeloid-Derived Suppressor Cells to Prevent Graft Rejection in Corneal Transplantation

Abstract

Myeloid-derived suppressor cells (MDSCs) are therapeutic agents to prevent graft rejection in organ transplants by modulating inflammation. Herein, the immunosuppressive effect of human cord blood MDSCs on corneal allograft models was confirmed. CB-MDSCs were locally (subconjuctival, 5 × 10⁵) or systemically (intravenous, 1 × 10⁶) injected twice on days 0 and 7. A corneal transplantation model was established using C57BL/6 and BALB/c mice, and corneal graft opacity was measured to evaluate graft rejection up to 6 weeks. Results showed that graft survival in the MDSCs groups increased compared to vehicle groups after 42 days. Systemic and local MDSC administration inhibited the maturation (MHC-II^hi CD11c+) of dendritic cells (DCs) and the differentiation of interferon γ+ CD4+ Th1 in draining lymph nodes (LNs). However, vehicle groups increased the infiltration of CD3+ T cells and F4/80+ macrophages and produced prominent neovascular and lymphatic vessels into the graft site with increased mRNA expression of VEGF-A/C and VEGFR-1/R-3. Local MDSCs administration showed prominent anti-angiogenic/anti-lymphangiogenic effects even at lower MDSCs doses. Thus, CB-MDSCs could relatively suppress the infiltration of pathological T cells/macrophages into the corneas and the migration of mature DCs into draining LNs Therefore, ocular and systemic MDSCs administration showed therapeutic potential for preventing corneal allograft rejection.

Keywords: Myeloid-derived suppressor cells (MDSCs); T cells; corneal transplantation; graft rejection; macrophages.

Figure 1

Comparison of corneal allograft survival according to the local and systemic administration of MDSCs. BALB/c (recipient) corneas were engrafted orthotopically onto C57BL/6 (donor) corneas by using PBSiv (n = 9), MDSCiv (n = 9), PBSscj (n = 11), and MDSCscj (n = 11). Locally and systemically injected MDSCs (scj, iv) groups displayed significantly better corneal allograft survival than PBS-injected (scj, iv) groups (* p < 0.05) for 42 days. Kaplan–Meier survival curves were obtained via the log-rank test for the statistical comparison of four groups.

Figure 2

Populations of major histocompatibility complex (MHC) II^hi CD11c+ cells and differentiation of interferon (IFN)-γ-expressing effector T cells in draining lymph nodes after corneal transplantation. Populations of MHC II^hi CD11c+ dendritic cells (DC) and differentiation of interferon (IFN)-γ-expressing effector T cells in draining lymph nodes (LNs). (A) Representative flow cytometry plot showing MHC II^hi CD11c+ cells after CD11c+ gating. (B) Representative flow cytometry plot presenting the differentiation of IFNγ+ CD4+ cells. (C) Quantitative analysis of MHC II^hi CD11c+ cell populations (n = 3). Comparison between MDSCs (MDSC iv, MDSC scj) and vehicle (PBSiv, PBSscj) showed that the MDSCs groups presented a decreased maturation of MHC II^hi CD11c+ cells, whereas the vehicle groups did not. The immunomodulating effect of MDSCs was significantly better in the local (scj) injection group than in the systemic (iv) injection group. (D) Quantitative analysis graph of IFNγ+ CD4+ cell differentiation (n = 3). MDSCs groups (MDSC iv, MDSC scj) led to inhibit differentiation of IFNγ+ CD4+ cells when compared with vehicle groups (PBSiv, PBSscj). Representative flow cytometric data from three independent trials with pooled cells from three mice per group. * p < 0.05, ** p < 0.01.

Figure 3

Immunohistochemical (IHC) staining of corneal allografts to evaluate infiltrating immune cells. (A) Representative IHC staining showed the naive group (A) and infiltration of CD3+ T cells (green; white arrow) and F4/80+ macrophages (red; white arrowhead) in the grafted corneas (B–E) on week 6 (20× magnification, scale bar = 50 µm). (F) Quantitative analysis of CD3+ T cell infiltration (n = 3). (G) Quantitative analysis of F4/80+ macrophages infiltration (n = 3). In the MDSCs groups, the infiltration of CD3+ T cells and F4/80+ macrophages in the grafted corneas was significantly suppressed regardless of the delivery site. Three to four different sections from three independent mice were randomly selected for counting blinded samples, and the average was calculated. Data were presented as average ± SEM. ** p < 0.01, *** p < 0.001.

Figure 4

Assessment of cellular apoptosis in the corneal graft 6 weeks after the operation. Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; green; white arrow) assay images (20× magnification, scale bar = 50 µm) of the corneal graft of each group; non-transplanted naive cornea (A), PBSiv (B), PBSscj (C), MDSCiv (D), and MDSCscj (E). (F) Quantitative analysis graph of TUNEL-positive apoptotic cells (green). MDSC-treated groups inhibited the cell death of the grafted corneas compared with that of the PBS-treated group (* p < 0.05, ** p < 0.01, *** p < 0.001). Each experiment consisted of three corneas per group.

Figure 5

Comparisons of neovascularization and lymphangiogenesis in corneal allografts via MDSC administration. Representative whole-mount corneal immunofluorescent CD31^hi (green) and LYVE-1^hi (red) staining images from each group: PBSiv (A), MDSCiv (C), PBSscj (B), and MDSCscj (D). The representative photograph is a combination of multiple stitched photographs (5× magnification, scale bar = 500 μm) taken after dividing the whole cornea into several parts. MDSC groups (MDSCiv, MDSCscj) presented the significantly suppressed areas of neovascularization (E), green-CD31^hi; white arrows for blood vessels and lymphangiogenesis ((F), red-LYVE-1^hi) rather than those of the vehicle groups (PBSiv, PBSscj). Data are presented as the mean ± SEM of three repeated experiments involving three corneas per group (E,F); * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

Real-time polymerase chain reaction analysis of the mRNA expression levels of angiogenesis and lymphangiogenesis on the grafted cornea. mRNA levels of angiogenesis ((A): VEGF-A and (C): VEGFR-1) and lymphangiogenesis ((B): VEGF-C, (D): VEGFR-3) on the grafted corneas 6 weeks after corneal transplantation. The MDSCs groups (MDSCiv, MDSCscj) were compared with PBSiv and PBSscj (n =  3–4, * p < 0.05, ** p < 0.01 and *** p < 0.001). The local and systemic administration of MDSC groups (MDSCiv, MDSCscj) showed a decreased mRNA expression of VEGF-A, VEGFR-1, VEGF-C, and VEGFR-3, in comparison with that of the PBS-treated groups (PBSiv, PBSscj). Data were normalized to GAPDH as internal control, and relative values were expressed as the fold change of the naïve corneas. Data are presented as mean ± SEM of three or four experiments. Each experiment consisted of three or four corneas per group.