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. 2018 Jan;37(1):95-101.
doi: 10.1097/ICO.0000000000001400.

Corneal Tissue From Dry Eye Donors Leads to Enhanced Graft Rejection

Affiliations

Corneal Tissue From Dry Eye Donors Leads to Enhanced Graft Rejection

Takenori Inomata et al. Cornea. 2018 Jan.

Abstract

Purpose: To assess the effect of dry eye disease (DED) in graft donors on dendritic cell (DC) maturation, host T-cell sensitization, and corneal allograft rejection.

Methods: Corneas of control (healthy donor) and DED mice (C57BL/6) were transplanted onto fully allogeneic naive BALB/c recipients (n = 10 mice/group). Long-term allograft survival was evaluated for 8 weeks. Corneas and draining lymph nodes (dLNs) were harvested at posttransplantation day 14 (n = 5 mice/group). The frequencies of MHCII CD11c DCs in the donor corneas and host dLNs and the frequencies of interferon (IFN)-γ and IL-17 CD4 T cells and Foxp3 expression by Tregs in host dLNs were investigated using flow cytometry. The enzyme-linked immunospot assay was used to assess host T-cell allosensitization through direct and indirect pathways (n = 3/group).

Results: Recipients of DED donor corneas showed significantly reduced graft survival (10%) compared with control mice (50% survival, P = 0.022), and had significantly increased frequencies of mature DCs in the grafted cornea (DED donor 44.0% ± 0.36% vs. healthy donor 35.4 ± 0.5%; P < 0.0001) and host dLNs (DED donor 25.1% ± 0.66% vs. healthy donor 13.7% ± 1.6%; P = 0.005). Frequencies of IFN-γ and IL-17 T cells were increased in the dLNs of recipients of DED corneas, whereas the expression (mean fluorescence intensity) of Foxp3 in Tregs was decreased significantly in these mice (DED donor 6004 ± 193 vs. healthy donor 6806 ± 81; P = 0.0002). Enzyme-linked immunospot analysis showed that the direct pathway of allosensitization was significantly amplified in recipients of grafts with DED (P = 0.0146).

Conclusions: Our results indicate that DED in the donor is a significant risk factor for subsequent corneal allograft rejection.

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Figures

Figure 1
Figure 1. Recipients of grafts with dry eye disease demonstrate reduced allograft survival
Corneal grafts were harvested from C57BL/6 mice with dry eye disease (DED) or from healthy C57BL/6 donors as the control. Corneal grafts were transplanted into healthy BALB/c host beds, and observed weekly over 8 weeks. (A) Kaplan-Meier survival curves showing graft survival of mice with healthy (control) vs. DED grafts (n=10/group, *p=0.0215); (B) Graft opacity scores were assessed using biomicroscopy (two-way ANOVA with Bonferroni post-test, n=10/group, ***p<0.0001).
Figure 2
Figure 2. Dry eye disease in donor promotes dendritic cell maturation in graft recipient
(A) Representative in vivo confocal microscopy (IVCM) images displaying dendritic cells in the cornea of mice with DED and in healthy donors (n=10/group). The size of image is 400 × 400 μm2. (B) Number of dendritic cells, identified as bright dendritiform cells, per section in DED and healthy donor corneas (***p <0.001). (C) Representative flow cytometry plots showing mature CD11c+ MHCIIhigh dendritic cells (DCs) in cornea 14 days post-transplantation. (D) Mean frequencies of mature CD11c+ MHCIIhigh DCs among CD45+ cells in the cornea were assessed using flow cytometry (n=5, ***p<0.001). (E) Representative flow cytometry plots showing mature CD11c+ MHCIIhigh DCs in the draining lymph nodes (dLNs). (F) Mean frequencies of mature CD11c+ MHCIIhigh DCs among CD45+ cells in the dLNs were assessed using flow cytometry (n=5, *p<0.05). p values were calculated using the Student’s t-test and error bars represent standard error of mean.
Figure 3
Figure 3. Dry eye disease in donor leads to increased Th1 and Th17 frequencies and decreased regulatory T (Treg) function
Host T cell responses in draining lymph nodes (dLNs) of transplant recipients was assessed 14 days post-transplantation using flow cytometry. (A) Representative flow cytometry plot showing CD4+ IFN-γ + Th1 cells. (B) Mean Th1 cell frequencies in the dLNs of recipients with healthy vs. dry eye donor corneas are shown (n=5, **p=0.0017). (C) Representative plots showing the frequencies of CD4+ IL-17A+ Th17 cells. (D) Mean Th17 cell frequencies in the dLNs of recipients with healthy vs. dry eye donor corneas are shown (n=5, p=0.0061). (E & F) The mean fluorescence intensity (MFI) of Foxp3 in Tregs from dLNs of recipients with healthy vs. dry eye donor corneas are shown, demonstrating significantly lower levels of Foxp3 expression by Tregs in recipients of grafts from dry eye donors (n=5, **p=0.0051). (G) The suppressive function of Tregs harvested from hosts with grafts derived from dry eye vs. normal healthy donors was compared using the T cell proliferation assay. Results show a modest, though statistically significant decrease in the suppressive function of Tregs in recipients of grafts from dry eye donors compared to healthy controls (54.8% vs. 63.1%, n=6, ***p<0.0001; p values were calculated using the Student’s t-test and error bars represent standard error of mean.
Figure 4
Figure 4. Dry eye disease in graft promotes host T cell allosensitization through the direct pathway
T cell allosensitization was assessed using the ELISPOT assay. The assay was carried out 14 days after transplantation to analyze CD4+ IFN-γ+ T cell sensitization through direct and indirect pathways. (A) Representative images of spots are shown (n=3). (B) Mean number of spots representing directly and indirectly allosensitized CD4+IFN-γ+ T cells in recipients grafted with healthy vs. dry eye donor corneas are shown (Direct pathway: *p=0.015; Indirect pathway: p=0.33; p values were calculated using the Student’s t-test and error bars represent standard error of mean.

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