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Tat Fong Ng, Isaac J Benque, Brandon S Lee, John Muus, Andrew W Taylor; Uveitic Retinal Pigment Epithelial cells do not suppress the phagocytic antigen processing pathways in antigen presenting cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):781. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal Pigment Epithelium (RPE) produce immune suppressive factors that regulate the functions of infiltrating leukocytes. In uveitis this regulation is diminished. Our previous results showed that factors produced by healthy RPE alter the phagocytic to antigen presentation pathway in macrophages that suppresses their ability to antigen-activate T cells. Here, we examined whether uveitic RPE can regulate the phagocytic pathway in antigen presenting cells (APC).
EAU was induced in C57BL/6 mice by injection of an emulsion of CFA and IRBP peptide. At the peak clinical score of EAU, the mice were sacrificed and eyes were collected. The anterior segment and the retina were removed. The RPE eyecup was incubated in serum free media for 24 hours and the conditioned media (CM) was collected. RPE eyecup from healthy mice served as control. The macrophage cell line RAW264.7 was treated with EAU-eyecup CM or Healthy-eyecup CM, and fed opsonized-Ovalbumin (OVA)-coated magnetic beads (Dynabeads) for 24 hours. The beads containing phagosomes were isolated using a magnet and were assayed by immunoblotting for Rab 5 (early endosome marker) and Lamp1 (marker of phagolysosome formation). To assay antigen-stimulating T cell activation, resting peritoneal macrophages were fed opsonized-OVA and treated with RPE eyecup CM for 24 hours, and the cells were washed and used as APC to stimulate CD4+ OVA-specific T cells. After 72 hours of incubation T cell proliferation was measured.
Phagosomes containing magnetic beads from EAU CM treated macrophages had significantly higher ratio of Rab5 expression to Lamp1 (1.2±0.13;n=4) from the Healthy CM (0.8±0.23;n=4). The macrophages treated with Healthy CM suppressed stimulated OVA-specific T cell proliferation (0.45±0.06%). In contrast, the EAU CM had no effect on macrophages stimulated proliferation of OVA-specific T cells (0.9±0.04%).
The results demonstrate that EAU RPE did not suppress the maturation of the phagosomes in macrophages and this was related to their ability to present antigen to activate effector T cells. These results suggest that one of the mechanisms of ocular immune privilege is to suppress the presentation of antigens within the ocular microenvironment, and that this regulation changes in disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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