Local Adoptive Transfer of Plasmacytoid Dendritic Cells as a Novel Therapeutic Approach for Corneal Nerve Regeneration

Abstract

Purpose : We have recently shown that plasmacytoid dendritic cells (pDCs) exert neurotrophic properties in the cornea. The aim of this study is to evaluate the therapeutic efficacy of local adoptive transfer of pDCs in corneal nerve regeneration.

Methods : Corneas of 6-8 week-old male wildtype (WT) C57BL/6 mice underwent deep stromal trephination with a 2mm trephine to sever corneal nerves. Splenic GFP⁺ pDCs from DPE-GFP×RAG1^-/- mice and WT CD11b⁺ myeloid cells were isolated. After trephination, 10⁴ pDCs, CD11b⁺ cells, or PBS control were locally applied onto the corneas using Tisseel tissue glue. On day 3, corneas underwent flow cytometry to assess protein expression of NGF. On day 14, corneas were stained for βIII-tubulin (pan-neuronal marker), CD45 (pan-leukocyte marker), and MHC-II (maturation marker). Total length of corneal nerves was quantified via NeuronJ and densities of MHC-II⁺ cells were measured by ImageJ. ANOVA with LSD post hoc test was used to assess statistical significance. p<0.05 was considered significant.

Results : Confocal microscopy confirmed successful transfer of GFP⁺ pDCs to both central (331.5±42.7 cells/mm²) and peripheral corneas (447.9±74.5) on day 1 following local application of pDCs. Flow cytometry showed a 1.4-fold increase in the density of NGF⁺ cells on day 3 following adoptive transfer of pDCs, compared with Tisseel-only control. One-time adoptive transfer of pDCs was accompanied by enhanced nerve regeneration on day 14 post-trephination in both the center (44.5±10.1 mm/mm²) and periphery (75.9±10.9) of corneas, compared with transfer of CD11b⁺ cells (24.9±11.7, p=0.02 in center and 47.7±8.2, p=0.002 in periphery) as well as Tisseel-only controls (22.2±6.3, p=0.005 in center and 62.3±4.0, p=0.04 in periphery). In corneas treated with local pDC transfer, we observed no significant increase in the density of MHC-II expressing leukocytes in the center (188.3±32.1 cells/mm² vs. 246.4±61.4 in Tisseel-only and 301.7±68.2 in CD11b⁺ cell-treated) or periphery (205.4±24.4 vs. 250.8±18.3 in Tisseel-only and 239.8±23.8 in CD11b⁺ cell-treated) compared with control groups (p>0.05), suggesting safety of local pDC adoptive transfer.

Conclusions : Local adoptive transfer of pDCs can enhance corneal nerve regeneration following nerve damage and may serve as a novel cell-based therapeutic approach to treat corneal nerve damage.

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