Fig. 8

Immunohistochemical evaluation of graft outcomes across divergent immune competence modalities of a–c immunodeficient (Nude TP), d–f immune suppressed immune-competent (NN TP IS) and g–i non-immune suppressed immune-competent (NN TP NO IS) RD rats. IHC panel to investigate immune cell responses, identify graft cells and their photoreceptor identity. Segmentation of each stained section represents corresponding marker colocalization of a, d, g Iba 1 and CD68 (microglia/macrophage markers), b, e, h Ku80+ (RO graft cells of human origin) and CD8+ (CTLs), c, f, i Recoverin (calcium channel protein in photoreceptors and cone bipolar cells), CD161 (natural killer lymphocytes). Both Nude TP and NN TP IS long term survival transplants (6.7 and 6.3 months post-surgery) show similar distribution and density of ramified microglia (a, d) (wherein the only CD68 immunoreactivity was found in vascular endothelial cells of the choroid and retinal blood vessels), b, e dense aggregations of graft cells are found in ONL, while both IS and non-IS grafts are devoid of CD8+ T-cells. c, f Recoverin positive photoreceptors of graft origin are brightly fluorescent and often aggregate as dense mini rosettes. Conversely, host recoverin positive cone bipolar cells stain dimly and are found in the inner nuclear layer (INL). No CD161+ NK cells are found in the subretinal space of either nude TP or NN TP IS RD retina sections. g–i Xenograft Rejection resulting from no immune suppression: NN TP NO IS: Outcome of hESC-RO graft in immune competent RD rat 14 days post-surgery. g Graft immune cell infiltrate consisting of double positive Iba1+/CD68+ microglia with characteristic ameboid activated morphology and h abundant CD8+ T-cells among densely packed cells of RO tissue graft, indicated by Ku80 human nuclear protein staining (green). i Absence of positive CD161 reveals no natural killer lymphocytes present in this rejected graft. Cells staining brightly with recoverin indicate developing photoreceptors among transplanted RO cells. Dotted rectangles indicate areas of enlargement. Scale bars = 100 µm for the overviews, 50 µm for the enlargements