Engineering of NK activating receptor ligands enhances immune compatibility of MHC-I−/− iPSC-derived β cells for cell therapy of type 1 diabetes

Background \u0026 Aim Induced pluripotent stem cells (iPSCs) represent a renewable source of pancreatic β cells and an ideal platform for production of genetically engineered cell therapies for type I diabetes (T1D). Nevertheless, autologous cell engraftment is strongly limited by autoimmune response in T1D, whereas transplantation of allogeneic iPSC-derived β cells, as well as donor islets, results in progressive CD8+ T cell-mediated allograft rejection. Although proposed cell therapy with MHC-I−/− iPSC-derived β cells may result in prevention of CD8+ T cell-mediated immune response, it could trigger missing-self recognition of natural killer (NK) cells, nullifying this strategy. In light of this, we aimed to inhibit NK lysis by modulation of NK activating ligands to enhance transplant compatibility of MHC-I−/− iPSC derivatives. Methods, Results \u0026 Conclusion Methods - Differentiation of iPSCs into β cells was achieved by in vitro protocol mimicking pancreas development. Expression of NK ligands during β cell differentiation was assayed by flow cytometry, IF and qRT-PCR. Gene editing was performed by using CRISPR/Cas9 system. Immunogenicity tests were set up by culturing target cells with CD8+ T or NK cells, at different target-effector ratios. NK activation markers were assayed by flow cytometry. Results - Performing cytotoxicity tests on wild type and MHC-I−/− cell lines revealed that MHC-I−/− cells efficienlty escape CD8+, but not NK lysis. We found that iPSC-derived β cells express high levels of B7H3 and moderate levels of DNAM-1-ligand CD155, whereas NKG2D-ligands MIC-A, MIC-B and RAET1E were significantly down-regulated on plasma membrane of terminally differentiated β cells. Supposing that such surface ligands could be involved in the amplification of NK activating signals following loss of self on target cells, we generated MHC-I-deprived B7H3−/−, CD155−/− and B7H3−/−/CD155−/− iPSCs. All three iPSC lines correctly differentiated into functional pancreatic β cells and interestingly appeared to significantly reduce NK lysis compared to MHC-I−/− ones. We also confirmed this dampened response by evaluating levels of NK activation markers, such as CD107a, TNFα and IFNγ. Conclusion - Our study suggests NK activating receptor ligands as target to make graft invisible to immune recognition, offering new perspectives for using iPSC-derived β cells as next generation cell source for T1D treatment.