Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II

Sandra Petrus-Reurer,Álvaro Plaza Reyes,Pankaj Kumar,Emma Lardner,Hammurabi Bartuma,Michael Chrobok,Evren Alici,Helen Kaipe,Nerges Winblad,Monica Aronsson,Laia Gorchs,Helder André,Anders Kvanta,Arnika Kathleen Wagner,Fredrik Lanner

doi:10.1016/j.stemcr.2020.02.006

Abstract

SummaryHuman embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

Highlights

Age-related macular degeneration (AMD) affects more than 180 million people globally and is the most common cause of blindness in industrialized countries among people over 60 years (Gehrs et al, 2006; Jonas et al, 2017)
We have previously reported that xenogeneic hESC-retinal pigment epithelial (RPE) may survive in the subretinal space of albino rabbits for extensive periods under intravitreal steroid immunosuppression, and similar findings were reported for allogeneic RPE derived from induced pluripotent stem cells transplanted to non-human primates (Petrus-Reurer et al, 2017; Plaza Reyes et al, 2016; Sugita et al, 2017)
B2M and CIITA Loci Edited by CRISPR/Cas9 to Obtain Single- and Double-Knockout Lines Anchoring of human leukocyte antigen class I (HLA-I) molecules to the cell membrane is dependent on the b2 microglobulin protein and is encoded by the B2M gene (Nathenson et al, 1981)

Summary

Introduction

Age-related macular degeneration (AMD) affects more than 180 million people globally and is the most common cause of blindness in industrialized countries among people over 60 years (Gehrs et al, 2006; Jonas et al, 2017). We have previously reported that xenogeneic hESC-RPEs may survive in the subretinal space of albino rabbits for extensive periods under intravitreal steroid immunosuppression, and similar findings were reported for allogeneic RPE derived from induced pluripotent stem cells (iPSCs) transplanted to non-human primates (Petrus-Reurer et al, 2017; Plaza Reyes et al, 2016; Sugita et al, 2017) When rejection occurs it typically involves both the innate immune system, which may activate recipient natural killer (NK) cells by lack of a cognate HLA-I (missing self) (Karre et al, 1986; Ljunggren and Karre, 1990), and the adaptive immune system where an antigen-presenting cell (APC) engulfs the donor cell, processes the mismatched HLA-I and HLA-II molecules into peptides, which in turn are presented and recognized by CD8+ cytotoxic and CD4+ helper T-cells via HLA-I and HLA-II molecules, respectively (indirect allorecognition). Donor cell antigens coupled to HLA-II molecules on the APC activate CD4+ T-cells that can trigger CD8+ T-cells, NK cells and antibody-producing B cells (Bradley et al, 2002; de Rham and Villard, 2011)

Results

Discussion

Conclusion