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Abstract：
Allogeneic immune rejection is a major barrier for the application of human pluripotent stem cells (hPSCs) in regenerative medicine. A broad spectrum of immune cells, including T cells, natural killer (NK) cells, and antigen-presenting cells, which either cause direct cell killing or constitute an immunogenic environment, are involved in allograft immune rejection. A strategy to protect donor cells from cytotoxicity while decreasing the secretion of inflammatory cytokines of lymphocytes is still lacking. Here, we engineered hPSCs with no surface expression of classical human leukocyte antigen (HLA) class I proteins via beta-2 microglobulin (B2M) knockout or biallelic knockin of HLA-G1 within the frame of endogenous B2M loci. Elimination of the surface expression of HLA class I proteins protected the engineered hPSCs from cytotoxicity mediated by T cells. However, this lack of surface expression also resulted in missing-self response and NK cell activation, which were largely compromised by expression of β2m-HLA-G1 fusion proteins. We also proved that the engineered β2m-HLA-G5 fusion proteins were soluble, secretable, and capable of safeguarding low immunogenic environments by lowering inflammatory cytokines secretion in allografts. Our current study reveals a novel strategy that may offer unique advantages to construct hypoimmunogenic hPSCs via the expression of membrane-bound and secreted β2m-HLA-G fusion proteins. These engineered hPSCs are expected to serve as an unlimited cell source for generating universally compatible \"off-the-shelf\" cell grafts in the future.
摘要：
同种异体免疫排斥是人类多能干细胞（hPSC）在再生医学中应用的主要障碍。广谱的免疫细胞，包括T细胞,自然杀伤（NK）细胞，和抗原呈递细胞，导致直接杀死细胞或构成免疫原性环境，参与同种异体移植免疫排斥反应。仍然缺乏保护供体细胞免受细胞毒性同时减少淋巴细胞的炎性细胞因子分泌的策略。这里,我们通过内源性B2M基因座框架内的β-2微球蛋白(B2M)敲除或HLA-G1双等位基因敲入,对hPSC进行工程改造,使其表面不表达经典人类白细胞抗原(HLA)I类蛋白。I类HLA蛋白的表面表达的消除保护工程化hPSC免受T细胞介导的细胞毒性。然而,这种表面表达的缺乏也导致了自身反应缺失和NK细胞活化，β2m-HLA-G1融合蛋白的表达在很大程度上受损。我们还证明了工程β2m-HLA-G5融合蛋白是可溶性的，可分泌的，并且能够通过降低同种异体移植物中的炎性细胞因子分泌来保护低免疫原性环境。我们目前的研究揭示了一种新策略，该策略可以通过表达膜结合和分泌的β2m-HLA-G融合蛋白来构建低免疫原性hPSC。这些工程化hPSC有望成为未来产生普遍兼容的“现成”细胞移植物的无限细胞来源。
