In vitro and in vivo immunologic characterization of human embryonic stem cell-derived retinal pigment epithelial cells

Abstract

Purpose : Allogeneic human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could serve to replace lost tissue in geographic atrophy (GA). Here we assess human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cell immunogenicity when challenged with T-cells and natural killer (NK) cells in vitro, as well as adaptive and innate immunoresponses in vivo.

Methods : Derivation of hESC-RPE was carried out in xeno-free and defined conditions using laminin-521. The derived cells were analyzed for immune markers by immunofluorescence and flow cytometry. Additionally, carboxyfluorescein succinimidyl ester (CFSE) proliferation assay and interferon (IFN)-gamma ELISA were performed after co-culture with purified T-cells both under stimulatory and non-stimulatory conditions; similarly, chromium release cytotoxicity assay was assessed after co-culture with freshly isolated and interleukin (IL)-2-activated NK cells. Suspensions of differentiated hESC-RPE were transplanted subretinally into the large-eyed rabbit model. Following rejection of transplanted cells, the presence of immune cells was analyzed by immunohistochemistry and the existence of human-specific antibodies in serum was evaluated by flow cytometry.

Results : Mature hESC-RPE expressed HLA-I whereas IFN-gamma stimulation led to up-regulation of HLA-II. When co-cultured with T-cells, hESC-RPE exhibited different responses depending on the cell numbers present, ranging from an immunosuppressive capability to triggering T-cell produced IFN-gamma. When co-cultured with NK cells, a hESC-RPE maturity-dependent cytotoxcity was observed. Subretinal xeno-transplants of hESC-RPE were frequently rejected through time despite the presence of local immunosuppression. Infiltrating cells exhibited markers positive for T-cells (CD3), NK cells (CD56) and macrophages (RAM11). Additionally, anti-human antibodies were present in the serum of transplanted rabbits and correlated with the degree of rejection.

Conclusions : hESC-RPE can induce both adaptive and innate immunoresponses in vitro and in vivo thus supporting the need to develop strategies able to overcome host-donor mismatch in non-autologous cell-based treatment for GA.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.