The Role of Corneal Plasmacytoid Dendritic Cells in Immune Sensitization after Corneal Transplantation

Abstract

Purpose : Corneal allograft rejection is induced by adaptive immune responses. The purpose of this study was to investigate the role of corneal plasmacytoid dendritic cells (pDCs) in mitigating allosensitization and inducing tolerance to corneal allografts.

Methods : Wild type (WT) BALB/c (H-2d) mice were used as donors. Transgenic BDCA-2-DTR mice on C57BL/6 (B6; H-2b) background, in which local pDCs can be selectively depleted with subconjunctival (s.cj.) diphtheria toxin (DT) injections were used as recipients. WT B6 mice were used as sham controls. DT (30 ng) was injected s.cj. one day before transplantation and was continued every other day for two weeks. Graft opacity was evaluated up to 2 weeks after transplantation using a standard scale. Graft infiltration of CD45+cells (pan-leukocyte marker), CD68+macrophages and CD4+T cells were measured using flow cytometry. Ipsilateral draining lymph nodes (dLN) were assessed for host allosensitization using enzyme-linked immunospot (ELISPOT) assay and frequencies of IFNγ+CD4+T cells, IL17+CD4+T cells and CD25 expression levels in CD4+CD25+Tregs using flow cytometry.

Results : Mean opacity scores were significantly higher in pDC-depleted mice compared to the sham controls at day 7 (3.7/5 vs. 1.33/5; p<0.001) and day 14 (4.6/5 vs 3.41/5; p=0.027) post-transplantation. Infiltration of CD45+cells in corneal grafts was significantly higher in the pDC-depleted group vs. controls at day 7 (46.3% vs. 28.6% of total graft cells; p<0.01), including CD68+macrophages (59.07% vs. 33.4%; p=0.0005) and CD3+CD4+T cells (9.93% vs. 2.5%, p<0.0001). ELISPOT analysis showed increased IFNγ+T cells in both direct (p=0.026) and indirect (p=0.016) pathways of allosensitization in the pDC-depleted group vs. controls. Flow cytometric analysis of dLNs showed increased frequencies of CD4+IFNγ+T cells (2.2% vs. 0.95%; p=0.007) and CD4+IL17+T cells (2.87% vs. 1.67%; p=0.046) in the pDC-depleted hosts vs. sham controls. There was a significant decrease in mean expression of CD25 among Tregs in the pDC-depleted group vs. sham controls (mean fluorescent intensity: 211 vs. 154, p=0.04).

Conclusions : Depletion of pDCs in the cornea results in increased host allosensitization and accelerated leukocyte graft infiltration. These results demonstrate the capacity of pDCs to mediate T cell responses, and provide valuable evidence for cell-mediated therapy to promote corneal transplant survival.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.