Jt Ernest

Human fetal retinal pigment epithelium suppresses the activation of CD4(+) and CD8(+) T-cells.

Abstract · Background: The suppressive effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human CD4+ and CD8+ T-cells was evaluated in vitro. · Methods: Pure populations of CD4+ and CD8+ T-cells were isolated from human peripheral blood-derived buffy coats by negative immunomagnetic selection. The purity of the cells was examined by flow cytometry using anti-CD3-FITC, anti-CD4-FITC, anti-CD8-PE, and anti-CD20-PE mAbs. HFRPE cells were isolated from fetal eyes and pure cultures were obtained. The effect of normal or IFN-γ-activated HFRPE cells at early (P3) or late (P6) passages on the activation of CD4+ and CD8+ T-cells was assessed in two different T-cell activation assays. In both activation models anti-CD3 mAb (OKT3) provided the antigen-specific signal. The secondary signal for the activation of CD4+ and CD8+ T-cells was provided with anti-CD18 mAb (TS1/18) and anti-CD28 mAb (9.3) in the first and the second assay respectively. Cross-linking of these soluble mAbs was performed with sheep-anti-mouse IgG-coated latex beads. The T-cell activation was determined by cell proliferation measured by [3H]thymidine incorporation. In each activation assay T-cells were incubated with HFRPE cells in a ratio of T-cells to HFRPE of 1:1 or 1:4. · Results: CD4+ and CD8+ T-cells were activated by cross-linking CD3 and CD18 in the first assay (CD3/CD18) and CD3 and CD28 in the second assay (CD3/CD28). In both assays HFRPE inhibited the activation of CD4+ and CD8+ T-cells. IFN-γ-activated HFRPE cells totally suppressed the T-cell activation at a 1:1 ratio. This suppressive effect was weaker at lower cell ratios. Some donor variation was observed in the inhibition at the lower cell ratios, especially for the inhibition of CD8+ T-cell activation with anti-CD3/CD18. The passaging of HFRPE cells did not alter their suppressive effect on CD4+ and CD8+ T-cells. · Conclusions: HFRPE cells suppressed the activation of both CD4+ and CD8+ T-cells in vitro. These findings suggest that RPE-induced immune suppression may play a significant role in maintaining immune privilege in the subretinal space.

A model for xenogenic immune response

Abstract Purpose: To develop a model for analyzing the immune response after xenogenic human fetal retinal pigment epithelium (HFRPE) transplantation. Materials and methods: Pure sheets of HFRPE cells were isolated and attached to poly (dl-lactide-co-glycolide) polymer films and HFRPE spheroids were formed. The spheroids were transplanted into the subretinal space of New Zealand albino rabbits and were observed for 5 months. Bare polymer films were transplanted into the subretinal space of Dutch Belted pigmented rabbits, serving as control. Results: The polymer film was bioegraded within 3 weeks in the subretinal space. No signs of inflammation in the retina or choroid were observed. The HFRPE spheroids were easily transplanted into the subretinal space. The immune response was followed with ophthalmoscopy. Light microscopy indicated a localized immune response in the transplanted area in which the retina and the choroid were infiltrated with immune cells. This infiltration was denser in the choroid. Conclusions: HFRPE spheroid transplantation may be utilized as a model for studying the xenogenic immune response after HFRPE transplantation. This model may also have applications in evaluating the role of immune suppressive agents in preventing rejection after HFRPE transplantation.