chair of medicine Leiden University Medical Center Leiden, Zuid-Holland, Netherlands
Abstract: Mismatches in HLA haplotypes between donors and recipients significantly increase the risk of graft failure due to immune rejection. While knocking out beta-2 microglobulin (B2M) is the current standard for preventing HLA class I surface expression and protecting stem cell-derived products from allogeneic rejection, the complete ablation of HLA-I molecules can impair natural killer (NK) cell “self” recognition and disrupt critical immune-regulatory interactions. To address these challenges, we developed a novel hypoimmunogenic induced pluripotent stem cell (iPSC) line by inserting the cytomegalovirus-derived US2 encoding sequence into the AAVS1 safe harbor locus. Comparison of the transcriptomic profiles of B2M-deficient and US2-modified iPSCs to their parental counterparts, revealed that US2 expression does not affect stemness markers, including SMAD2, SMAD4, NANOG and SOX2, or differentiation potential into endothelial cells (ECs) or kidney organoids, as confirmed by lineage-specific marker expression. Flow cytometry (FACS) analysis revealed that US2-expression abrogates HLA-A2 cell surface expression while retaining detectable levels of non-classical HLA molecules, measured at 10- to 15-fold lower than control levels. Co-culture assays revealed that US2 expression prevented HLA-A2 alloreactive T cells activation, as shown by the absence of difference in MIP-1β secretion compared to control. Moreover, US2 expression significantly reduced NK cell activation, with CD107a degranulation reduced by 5% and MIP-1β secretion by 20% to 40% compared to B2M KO ECs. These findings establish US2-mediated "stealth" technology as a refined alternative to B2M knockout, offering selective modulation of HLA expression to mitigate immune rejection while preserving critical immune-regulatory interactions.
Funding Source: This study was funded by LUF/Stichting Prof. Jaap de Graeff-Lingling Wiyadharma Fonds 2020-01 and the Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW, supported by Novo Nordisk Foundation grants (NNF21CC0073729)).
