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Zala Luznik, Andreja Nataša Kopitar, Alojz Ihan, Marko Hawlina, Petra Schollmayer; Characterization of dendritic cell subtypes in native and cultured cadaveric human limbal tissue on amniotic membrane. Invest. Ophthalmol. Vis. Sci. 2017;58(8):982.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal allograft rejection is the leading cause of graft failure. Resident dendritic cell (DC) maturation plays a critical role in the initiation of host allosensitization and graft rejection. There are two developmental lineages of DC: a myeloid (mDC) and a lymphoid (pDC) lineage, with different biological properties. The distribution of DC subtypes in whole corneal tissue and during limbal tissue cultivation is still not well determined. Thus, we hypothesized that a difference in DC content would be observed between non-cultured human cadaveric limbal tissue (control) compared to limbal explant cultures on amniotic membrane (AM). Secondly, we aimed to analyze the distribution of DC subtypes in whole preserved human cadaveric corneal tissue divided into central corneal, superior, inferior, nasal and temporal peripheral corneolimbal areas.
The expression of CD11c (for mDC) and CD303/CD123 (for pDC) was evaluated by flow cytometry on limbal explant cultures cultivated on either the epithelial or stromal side of the AM and compared with directly isolated cells from cadaveric whole corneoscleral tissue, divided into specific areas for comparison. Additionally, the expression of co-stimulatory molecules CD80, CD86, and activation markers HLA-DR, CD83 was investigated, as well as the expression of corneal epithelium marker CK12 and ABCB5, a new epithelial stem cell marker. Student’s t-test was used for statistical analysis.
Expression of pDC markers (CD303+/CD123+:10.2±1.0% vs 3.9±0.3%), mDC (CD11c+:7.0 ±0.9% vs 4.0±0.4%) and stem cell ABCB5 marker (26.4±1.6% vs 9.5±1.1%) was significantly higher (p < 0.05) in native (non-cultured) cadaveric corneolimbal tissue (N=15) compared to limbal explant cultures cultivated on AM (N=6). Cells positive for pDC and mDC markers were found in all examined corneolimbal areas (central 7.5 mm corneal > inferior > nasal > superior, and temporal limbus) with a prevalence of pDC (p < 0.05), but with no statistically significant difference between activated pDC and mDC. In contrast, in limbal explant cultures the percentage of pDC and mDC was similar, with no statistically observed differences between both sides of AM.
The DC content was significantly lower after ex vivo limbal explant cultivation, which is consistent with our hypothesis. DC subtypes and ABCB5 positive cells were found in all tested corneolimbal areas.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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