Sulfo-NHS-SS-Biotin RPE cell associated allografts have been

RPE cell-associated allografts have been considered for the treatment of ocular diseases such as age-related macular degeneration (AMD). We successfully established human RPE Sulfo-NHS-SS-Biotin from human iPSCs (Kamao et al., 2014; Sugita et al., 2015). In addition, we recently transplanted an iPSC-derived RPE (iPS-RPE) sheet into an AMD patient autograft. RPE cells including iPS-RPE cells have immunosuppressive properties; human RPE cells suppress T cell activation and can convert T cells to regulatory T cells (Horie et al., 2010; Imai et al., 2012; Sugita et al., 2015; Usui et al., 2008). However, several groups in human clinical trials found that RPE allografts did not survive because of immune rejection (Algvere, 1997; Algvere et al., 1999; Peyman et al., 1991; Weisz et al., 1999). Algvere et al. (1999) reported that immune rejection after RPE transplantation in humans includes loss of visual function over the transplant, development of an exudative response (e.g., serous retinal detachment), fluorescein leakage of the grafts, disruption of the grafts, depigmentation of the grafts, and encapsulation of the grafts. However, there have been no previous reports of how antigen and cell type affect the outcome of the retinal transplantation. In addition, as far as we know, no one has reported that RPE cells derived from embryonic stem cells (ESCs)/iPSCs are recognized by MHC-restricted immune cells, especially T cells.
Results
Discussion In the present study, we prepared human RPE cells derived from iPSCs as stimulators and T cells as responders to evaluate immune rejection in vitro. The ability of RPE cells to stimulate the bystander T cells was analyzed using MLR and IFN-γ production by T cells. In some experiments, APCs and anti-CD3 antibody (first signal blocking) were also used for the in vitro rejection assay. For the assay, we used T cell culture medium containing recombinant human IL-2. IL-2 is a growth factor for all subpopulations of T cells and acts as an antigen-nonspecific factor in the proliferation of these T cells. Th1 cells produce IL-2 as well as IFN-γ. The MLR assay is a useful tool with which to investigate the mechanisms of allogeneic responsiveness in vitro (allo-MLR) including the response to RPE cells (Kamao et al., 2014). In addition, we purified CD4+ T cells co-cultured with iPS-RPE cells in the presence of recombinant IL-2. To evaluate the recognition of T cells, we collected the supernatants to measure inflammatory cytokines such as IFN-γ for CD4+ T cells. We then analyzed whether the T cell recognition of RPE cells was HLA restricted. In this study, we focused on HLA-A, -B, and -DRB1 among the six HLA loci antigens, although we tested all six HLA genotypes, A, B, C, DRB1, DQB1, and DPB1, in PBMCs and RPE cells. Retinal allografts have been considered for the treatment of ocular disease, and there is extensive evidence that allografts can survive in the retina without immunosuppression (Algvere et al., 1999). However, we previously showed that allogeneic transplants of iPS-RPE cells in animal models could elicit immune responses in the retina (Kamao et al., 2014), and several groups found that RPE allografts did not survive in human clinical trials (Algvere, 1997; Algvere et al., 1999; Peyman et al., 1991; Weisz et al., 1999). There have been no previous reports of how antigen (including MHC antigen) and cell type affect the outcome of retinal transplantation. In humans, allogeneic cell transplants almost always include a short course of immunosuppression, commonly cyclosporine A or tacrolimus, to minimize the chances of graft rejection through a T cell-mediated adaptive immune response (Clipstone and Crabtree, 1992). Eventually we might be able to control immune rejection by using T cell-specific medication. In addition, in immunohistochemistry with retinal sections, many T cells had invaded the retina after transplantation of iPS-RPE cells/sheets (allografts) in animal models, whereas control retinal sections examined by immunohistochemistry had no T cells in the retina (our unpublished data). Therefore, the T cell-mediated immune response plays a critical role in the pathogenesis of immune rejection after allogeneic RPE transplantation. As revealed in the present study, T cells exposed to iPS-RPE cells exhibited a Th1-type response (IFN-γ+ IL-2+ IL-1β+ T-bet+ STAT1+ CXCR3+). In fact, RPE cells, especially IFN-γ-treated RPE cells, can produce Th1-related chemokines (IFN-γ-related chemokines) such as CXCL9, CXCL10, and CXCL11 (Juel et al., 2012).