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G. M. Frank, R. L. Hendricks; In situ Ablation Strategy of Corneal CD11c+ cells in Herpes Stromal Keratitis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2645.
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Herpes Stromal Keratitis (HSK) is an immunopathological disease of the cornea. HSK is characterized by recurrent bouts of inflammation, associated with leukocytic infiltration and corneal scarring. In murine models, CD4+ T cells orchestrate HSK. Several studies have suggested corneal dendritic cells (DC) have a vital role in stimulating CD4+ T cells within the cornea. In skin models, dermal DC do not directly present antigen, instead transporting it draining lymph nodes. The direct or indirect role of resident corneal DC in presenting HSV-1 antigens requires clarification. Here we describe a method to deplete DC in the cornea without disrupting lymphoid -resident DC populations.
CD11c-DTR mice, with CD11c promoter driven expression of EGFP and the diphtheria toxin (DT) received HSV-1 corneal infections. Subconjunctival (sc) or intraperitoneal (ip) injections of DT were given 3 days post infection. Flat mounts of corneas and dispersed draining lymph nodes (DLN) were prepared at various times after DT treatment. Corneas were stained for CD11b and MHC II and examined by confocal microscopy. DLN cells were stained for CD11c, CD11b, and MHC II and analyzed by flow cytometry.
In non-infected corneas CD11c+ cells were readily detected via EGFP expression, and were depleted by sc injections of >20 ng of DT. CD11b+ cells were slightly increased, suggesting a role in removing DC debris. In contrast, DC depletion from infected corneas required sc injections of ≥ 50 ng of DT. Sc DT treatments did not influence DC populations in the DLN. The DC were efficiently depleted from both the cornea and DLN by ip DT treatments ≥ 150 ng.
Our findings establish the value of this model for depleting cornea resident DC without influencing the DLN resident DC. We further establish that DC can be locally depleted from infected corneas where there is constant replenishment from the blood without influencing the local macrophage population. We are currently using this approach to investigate the relative role of corneal and DLN DC for activating naïve HSV-specific CD4+ T cells in DLN and restimulating CD4+ effector T cells in the infected cornea.
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